CN1753995A

CN1753995A - Precise breeding

Info

Publication number: CN1753995A
Application number: CN 03808643
Authority: CN
Inventors: C·M·T·罗门斯; J·叶; H·颜; K·M·M·斯沃尔兹; J·梅嫩德斯－胡马拉; L·W·布林克霍夫; C·里切尔
Original assignee: JR Simplot Co
Current assignee: JR Simplot Co
Priority date: 2002-02-20
Filing date: 2003-02-20
Publication date: 2006-03-29

Abstract

The present invention relates to a new plant breeding process. The process improves the agronomic performance of crop plants by using genetic material that is also used in classical breeding. Instead of sexually recombining entire genomes at random, as is done in classical breeding, specific genetic elements are rearranged in vitro and inserted back into individual plant cells. Plants obtained through this new plant breeding process do not contain foreign nucleic acid but only contain nucleic acid from the plant species selected for transformation or plants that are sexually compatible with the selected plant species. Plants developed through this new plant breeding process are provided. In particular, potato plants displaying improved tuber storage and health characteristics are provided.

Description

Precise breeding

Invention field

The application requires the right of priority of U.S. Provisional Application sequence number 60/357,661 and 60/377,602, and it is incorporated by reference here.The present invention relates to improve the method for nutrition, health and the agronomic characteristics of plant by DNA special, that know in the modified plant genome.Opposite with traditional plant breeding, method of the present invention not can with the unknown or be incorporated into during plant genetic forms by the gene of genotoxic potential.In addition, method of the present invention is different with conventional genetic engineering strategy, not with the allos species, promptly can not with will by the plant that genetic engineering is modified alternately can be pregnant the nucleic acid of plant be incorporated in the genome of plant.Plant by plant breeding method exploitation of the present invention shows the agronomic characteristics that improves.The particularly preferred plant of the present invention comprises that demonstration has improved the potato of healthy and stem tuber storage characteristic, and shows the turfgrass that has improved disease and drought tolerance.

Background

Typically be to improve the agronomic characteristics of plant by traditional plant breeding or genetic engineering always.Traditional breeding generally causes the nucleic acid of the unknown is transferred to another kind of plant from a kind of plant.Gene engineering is incorporated into heterologous nucleic acids in the Plant Genome, promptly DNA be not to derive from plant or be not derive from will by the natural plant that genetic engineering is modified alternately can be pregnant plant.For example, genetic engineering is incorporated into non-plant nucleic acid in the Plant Genome.Traditional breeding and genetic engineering strategy both produce contains the Plant Genome of not expecting with unwanted genetic material, and the plant hybridization that obtains or genetically modified shows disadvantageous characteristic.The defective that can prove these two kinds of strategies is deleterious to transgenic plant, also is deleterious to the animal and human who eats these products.

Traditional breeding method depends on the transfer of unknown DNA

Plant breeding generally relies on recombinating at random of plant chromosome to have new and the mutation that improves characteristic with generation.Therefore, by numerous filial generations that screening obtains from plant hybridization, the breeder can identify those and show desired characteristic, and for example output increase, vigor enhancing, disease and insect-resistant improve or viability enhanced plant under drought condition.Yet traditional breeding method is an effort and consuming time, and the general only demonstration of the new mutation improvement of appropriateness relatively.

In addition, traditional plant breeding generally causes various unknown genes is transferred in the Plant Genome.The deleterious allergen of in the gene of those transfers some codings potential probably, for example potato tuber albumen, lectin, chitinase, proteolytic enzyme, thaumatin (thaumatin) sample albumen, fat transfer protein, amylase, trypsin inhibitor, and seed storage protein (Breiteneder etc., J Allergy Clin Immunol 106:27-36).

Similarly, may in the biosynthesizing of toxin, relate to gradually osmotin gene, wherein toxin comprises mountain black pigment used by women in ancient times to paint their eyebrows legumin (lathyrogens), hydrazine, glucosinolate and goitrogen, tonka bean camphor, saponin(e, alkaloid, glycoalkaloid, biogenic amine, enzyme inhibitors, as lectin (hemagglutinin), trypsin inhibitor, huge legendary turtle is closed substrate such as phytinic acid and oxalic acid, ribose toxin (ribotoxins), antimicrobial peptide, amino acid such as β-N-oxamido--L-L-Ala, atractyloside, oleandrine, taxol, and isoquinoline 99.9 (Pokorny, Cas Lek Cesk136:267-70,1997).Wild crop relatives by making great efforts to be used as before " exploitation " food consumption genetic diversity, risk (the Hoisington etc. that unexpectedly these poisonous substances are incorporated in the humans and animals provand product have further been improved, Proc Natl Acad SciUSA 96:5937-43,1999).

Although traditional plant breeding can easily be incorporated into the gene that relates in food crop and the plant in the anti-nutritious cpds of not expecting, it can not easily be removed them.For example, spent about 15 years to reduce deleterious phytate level (Raboy, J Nutr 132:503S-505S, 2002) in cereal and the paddy rice by deactivation Lpa gene.Realize that with long time frame positive result is unpractiaca, particularly owing to need more effectively and efficiently to improve the food crop method for quality now forwardly.Find just that recently an example with the synthetic relevant gene of anti-nutritious cpds is polyphenoloxidase (PPO) gene, some phenolic compound of its oxidation produces mutagenic, the carcinogenic and cytotoxic reagent (Kagan etc. that resemble phenoxy group free radical and the quinone derivative, Biochemistry 33:9651-60,1994).The multiple copy that in the genome of plant such as potato, has this gene, the feasible activity that is difficult to reduce PPO especially by breeding.

If their hereditary basis is known seldom or is known nothing, so even need more times to remove anti-nutritious cpds.For example, never gene and the accumulation that is heated to 160 ℃ or potato middle and high concentration acrylamide when higher at some are interrelated, acrylamide is a kind of strong neurotoxin and mutagenic compound (Tareke etc., J Agric Food Chem.50:4998-5006,2002).Therefore during the processing of using traditional breeding method, be difficult to develop efficiently the new potato mutation that can produce less acrylamide.Therefore, need plantation to have this class hazardous compound of lower level, but do not use the potato of the unknown or heterologous nucleic acids to be rich in the food of carbohydrate, for example wheat with other.

Can during processing, accumulate and be difficult to reduce to other anti-nutritious cpds minimum or that eliminate and comprise Maillard reaction (Maillard-reaction) product N-nitroso-group-N-(3-ketone-1 by breeding, the 2-butyleneglycol)-3 '-nitro tyrasamine (Wang etc., Arch Toxixol70:10-5,1995), and 5 hydroxymethyl 2 furaldehyde (Janzowski etc., Food ChemToxicol 38:801-9,2000).Never the also known demonstration slander of the other maillard reaction product that is characterized well becomes characteristic (Shibamoto, Prog Clin Biol Res304:359-76,1989).

Because traditional plant breeding inherent out of true, the level that promptly improve nutritious cpds useful in the food crop may be difficult equally.For example, expectation improves the level (Topping etc., Physiol Rev 81:1031-64,2001) of " Resistant starch " in the various crops.This starch finally is responsible for promoting immunne response, suppresses the potential pathogenic agent, and the sickness rate (Brid etc., Curr Issues IntestMicrobiol 1:25-37,2000) that reduces the disease that comprises colorectal carcinoma.Yet the plant that only available Resistant starch level increases is to resemble maize mutant strain " amylose extender ", " dull " and " sugary-2 " such low-producing mutation.New high-resistance starch source can be attached to this sanatory composition in the diet more widely as the generation of potato.

The genotype that can not handle plant safely often causes using outside pharmaceutical chemicals to induce the genotype of expectation.Although numerous procedures of breeding postpones stem tuber germination, yet for example, can't buy without sprout inhibitor and handle the potato mutation that just can preserve the several months.The latter, relevant as sec.-propyl-N-chloro-phenyl--carbamate (CIPC) with acute toxicity and tumor development, and may be present in the potato food of processing with the concentration between 1mg/kg and the 5mg/kg.

Genetic engineering depends on the transfer of foreign DNA

Genetic engineering can be used to modify, produce or remove some characteristic of plant.Although have limited progress aspect nutritive value that improves plant and the health characteristics always, the improvement of most target plant characteristics has promoted easy raise crop.Therefore, certain plants is resisted careless glycosides phosphine weedicide, because they contain 5-enol pyruvoyl shikimic acid-3-phosphate synthase gene (Padgette etc., Arch Biochem Biophys.258:564-73,1987) of bacterium.Similarly, anti-insect, antiviral and antimycotic botanical variety (Shah etc., Trends in Biotechnology 13:362-368,1995 have been produced in genetic engineering; Gao etc., Nat Biotechnol.18:1307-10,2000; Osusky etc., Nat Biotechnol.18:1162-6,2000), but almost there is not a kind of benefit that strengthens nutrition or health that has.

According to the known technology of standard, the gene " expression cassette " that will comprise gene and regulatory element is inserted in the edge sequence of the isolating transfer DNA s of edaphic bacillus (" T-DNAs ") and is incorporated into the Plant Genome.Therefore, the transfer of agrobacterium-mediated T-DNA material comprises following standard step classically: the vitro recombination of (1) genetic elements, wherein at least one element is allogenic, the expression cassette that is used for transformation and selection with production, (2) usually this expression cassette is inserted into the T-DNA district of two carriers with at least a other expression cassette that contains foreign DNA, it is that the hundreds of base pair of the edaphic bacillus DNA of T-DNA edge sequence constitutes by both sides usually, (3) will be in the sequence between the T-DNA edge, some or all that usually follow other two carrier sequences together, (Agrobacterium) transfers to the vegetable cell from edaphic bacillus, and the vegetable cell of (4) screening stable conversion.Referring to, for example U.S. Patent number 4,658, and 082,6,051,757,6,258,999,5,453,367,5,767,368,6,403,865,5,629,183,5,464,763,6,201,169,5,990,387,4,693,976,5,886,244,5,221,623,5,736,369,4,940,838,6,153,812,6,100,447,6,140,553,6,051,757,5,731,179,5,149,645, with EP 0120,516, EP 0257,472, EP 0 561, and 082,1,009,842A1,0853,675A1,0486,233B1,0554,273A1,0270,822A1,0174,166A1 and WO 01/25459.

Therefore, genetic engineering method depends on exogenous nucleic acid is incorporated in the provand.Those technology will be by several in the fusion transfer plant transformed kind more than 20 the complexity that isolating extrinsic genetic elements is formed from virus, bacterium and plant.Such external source element comprises regulatory element such as promotor and terminator, and in the expression of new features, relate to or identify or the gene of selection markers as transformation event.Although will check the safety of the food that contains foreign DNA before regulatory approved, many human consumers still are concerned about the remote effect of the edible food of expressing foreign protein, and this foreign protein is to produce by the gene that obtains from other non-plant kind.

A regulatory element that generally uses is 35S " super " promotor of cauliflower mosaic virus (CaMY), and it generally is used to induce with it direct-connected genetically modified high level expression in plant engineering.Yet this 35S promoter also can improve near the expression (Weigel etc., Plant Physiol., 122:1003-13,2000) of the natural gene it.This class promotor may be therefore in the expression of native gene slander lead unpredictable change, may cause ill effect, as increasing alkaloid yield.Preferred " by force " promotor those isolating promotors from virus normally, these viruses are as paddy rice tungro baculovirus, corn streak virus, cassava vein virus, Mirabilis jalapa virus, peanut chlorotic streak cauliflower mosaic virus, figwort mosaic virus and chlorella virus.The clone is from the bacterium kind for other promotor of often using, and the promotor that comprises nopaline synthase and octopine synthase gene.

In order to reach the suitable termination of genetic transcription, the terminator sequence is fused to the genetic elements that genetically modified 3 '-end and terminator sequence comprise the nopaline synthetic enzyme and the octopine synthase gene of edaphic bacillus.Can use other genetic elements to come further reinforcing gene expression or with some cell cell of expressed proteins target.These elements comprise the intron that promotes transgene expression and with the signal peptide sequence of some cell cell of foreign gene target, usually from the external source plant species.

Some related gene the most often comes from the allos source in the expression of new features.If the use natural gene, so often with them oppositely in transgenic plant, making this expression of gene silence, and with foreign DNA such as selected marker's cotransformation.The main drawback of this " antisense " technology is that reverse DNA contains the new and open reading-frame (ORF) that do not characterize that inserts usually between promotor and terminator.Therefore, all are with deriving from starch genes involved RI (Kossmann etc., United States Patent (USP) 6,207,880), L-and H-type glucosan phosphorylase gene (Kawchuk etc., United States Patent (USP) 5,998,701,1999), polyphenol oxidase genes (Steffens, United States Patent (USP) 6,160,204,2000) and Q-enzyme I and II gene (Schwall etc., the potato plant of antisense construct thing genetic modification NatureBiotechnology 18:551-554,2000) is all expressed the new peptide that is made of at least 50 amino acid (table 1) potentially.These new peptides may hinder development of plants and/or reduce the nutritive value of potato, thereby are unwelcome.

Be incorporated into the marker gene of routine in the genetic constructs and be used to select transformation event.They are given and transform bouvardin resistance or Herbicid resistant (U.S. Patent No. 6,174,724), metabolism advantage (U.S. Patent No. 5,767,378) or paramophia phenotype (U.S. Patent No. 5,965,791).This class marker is generally from bacterial origin.

In addition, because that T-DNA shifts is disloyal, about 75% transformation event also contains plasmid " main chain " sequence (Kononov etc., Plant J.11:945-57,1997) except containing T-DNA in plant such as tomato, tobacco and potato.The existence of this main chain sequence is unwelcome because they be external source and generally contain replication orgin and antibiotics resistance gene mark.

Really exist and remove the whole bag of tricks that resembles element the allos marker gene, a kind ofly can be applicable to genetically engineered plant at an easy rate but seldom have.According to a kind of such method, the gene or the nucleotide sequence of marker gene and expectation placed on the different carriers.With the single edaphic bacillus bacterial strain (U.S. Patent No. 6 that contains two carriers, 265,638) or with two edaphic bacillus bacterial strains, wherein each bacterial strain carries in two carriers one, may cause uncorrelated integration incident accidentally in order to infected plant, it can carry out heredity by outbreeding and separate.The main drawback of this method is that the heredity separation of locus may be to require great effort very much with consuming time, if particularly link together with T-DNA integration incident.In addition, this method can not be used in agamic plant widely, this plant vegetative propagation, as the Kentucky bluegrass, the perhaps crop of vegetative reproduction such as potato, because inbreeding depression, high-caliber heterozygosity and low reproductivity level, it can not be cultivated at an easy rate.

Another method of removing the allogeneic heredity element relies on foreign gene such as selected marker, is inserted in the transposable element.The transposable element of this modification may then be gone out by montage from genome with low frequency.Then must carry out traditional hybridization so that the element of swivel base is separated (U.S. Patent No. 5,482,852) with the host with unconverted plant.As described in former method, this selectable method can not be used for vegetative reproduction or agamic botanical system.

Remove the third method of marker gene and use Cre/lox locus specificity recombination system (the Dale ﹠amp of P1 phage; Ow, Pro.Natl.Acad.Sci.USA, 88:10558-62,1991).Between two lox sites marker gene is being inserted with Cre recombinase gene and the mosaic gene (two genes all have their promotor and terminator) that relates in the inducing of Cre, cause the zone (Zuo etc. that excision is described by the lox site during regenerative process, Nat.Biotechnol., 19:157-61,2001).The process of this complexity is that efficient is low and insecure, and may cause genomic instability.

Nearest research has reported that some plant genes itself can be used as transformation marker.The example of this class plant mark comprises Pga22 (Zuo etc., Curr Opin Biotechnol.13:173-80,2002), Ckil (Kakimoto, Science 274:982-985,1996) and Esrl (Banno etc., Plant Cell 13:2609-18,2001).Yet all these genes all cause the phytokinin reaction, and this has given transgenic plant bad phenotype.In addition, in case, just will still need to remove this class plant mark by any conversion the in the aforesaid method.

The selectable method that transforms plant also is based on the vitro recombination of foreign genetic element, and depends on the bacterial plasmid sequence that is used to maintain in intestinal bacteria (E.coli), and partial sequence is wherein integrated in conversion process altogether.The example that transforms these class methods of plant with foreign DNA is depicted in United States Patent(USP) Nos. 5,591,616,6,051,757,4,945,050,6,143,949,4,743,548,5,302,523 and 5,284,253.

Also may obtain unmarked transgenic plant by omitting the preceding any screening step of regeneration.The shortcoming of this method is that the most incidents that produce by this method will be represented unconverted plant or chimeric plant, because they are not to derive from single plant transformed cell usually.Using the step of unmarked thing to identify all to contain in all cells identical DNA to insert segmental transgenic plant is extremely difficult and effort.

Therefore, need improving plant, to make it surpass the plant that can obtain by traditional breeding cross and conventional genetic engineering be very important, its do not rely on the unknown or exogenous nucleic acid be inserted in the Plant Genome.Correspondingly, the invention provides the method and composition of the genetic stocks that is used for accurate modified plant self.Therefore, " precise breeding " of invention strategy is not induced unwelcome phenotype and not with in nucleic acid introduced plant genome the unknown or external source.

General introduction

The invention provides that heredity ground improves the nutritive value of plant and agronomic characteristics and not with the unknown or foreign DNA for good and all or stably be incorporated into method in this Plant Genome.The method according to this invention, from the plant species of expectation or from the affine plant species of vegetalitas of expectation separate nucleic acid, gene element and gene specific, that know, through modifying, then turn back to again in the genome of desired plant species.This modification may need the isolated nucleic acid sequences of suddenling change, the deletion isolating nucleic acid partial sequence, perhaps simply isolating nucleic acid is connected on another polynucleotide, for example with isolating nucleic acid subclone in plasmid vector.

Correspondingly, the transgenic plant that produce by methodology of the present invention do not have the genome that comprises any external source species nucleic acid.Therefore, its genome of transgenic plant that method of the present invention is produced does not comprise the promotor of non-plant kind, the terminator that does not comprise the non-plant kind, the 5 '-non-translational region that does not comprise the non-plant kind, the 3 '-non-translational region that does not comprise the non-plant kind does not comprise the marker gene of non-plant kind, does not comprise the regulatory element of non-plant kind, the gene that does not comprise the non-plant kind, and do not comprise any other polynucleotide that from the genome of non-plant kind, obtain.

Therefore, the invention provides-kind produce the method for stable transgenic plant, this plant shows not by the modification phenotype of non-conversion plant performance, comprises (a) polynucleotide transformed plant cells with expectation; (b) growing plant from cell transformed; (c) screening is with the plant of the new phenotype of demonstration of the polynucleotide stable conversion of described expectation, and wherein the plant that grows from corresponding non-transformed plant cells does not show this new phenotype.Preferably, the polynucleotide of expectation are made of following sequence basically: (i) nucleotide sequence, it separates certainly and/or the natural vegetable cell genome that belongs to, or belong to other plant mutually of the same race, or separate from and/or the natural genome that belongs to the affine plant species of the vegetalitas of originating with isolating vegetable cell; (ii) at least a dna sequence dna, it is an edge sample sequence, it has a sequence, natural genome or the natural genome that belongs to phase kindred plant cell that belongs to described vegetable cell of this sequence, the perhaps natural sequence that belongs to the affine plant of vegetalitas of originating with isolating vegetable cell, wherein sample sequence in edge can stably be incorporated into the polynucleotide of expectation in the genome of described vegetable cell.

Preferable methods of the present invention need produce demonstration not by the transgenic plant of the non-conversion modification phenotype that plant showed, comprise that (a) infects explant with edaphic bacillus, this edaphic bacillus (Agrobacterium) has (i) a kind of " P-DNA " ' carrier, it contains the natural polynucleotide that have of transgenic plant and (ii) a kind of " LifeSupport " carrier that comprises selected marker's expression cassette that contains of expectation; (b) time of selection selected marker transient expression, preferred 1-10 days, 3-7 days, perhaps 4-5 days; (c) explant is transferred in the regeneration culture medium to allow to form spray; (d) the numerous sprays of screening comprise at least one copy of the polynucleotide of expectation to determine which spray in its genome, and which branch does not contain any exogenous nucleic acid in its genome in those sprays, as the selected marker; (e) allow in its genome, to contain the polynucleotide of expectation and the spray that do not contain any marker gene DNA grows up to whole plant, wherein resulting whole plants shows modifies phenotype, and this phenotype is not shown by the plant that grows from non-transformed plant cells mutually of the same race.

According to such method, the polynucleotide of expectation (i) only constitute by separating oneself and/or the natural genomic element that belongs to vegetable cell kind or the affine kind of its property basically; (ii) comprise at least a edge member, this element has a separation oneself or natural vegetable cell kind or the genomic sequence of the affine kind of its property of belonging to, and this sequence can stably be incorporated into the polynucleotide of expectation in the genome of the vegetable cell that is exposed to carrier; Be incorporated in the genome that transforms plant with (iii) being stabilized; Wherein this method is not incorporated into non-plant kind or foreign DNA in the genome that transforms plant.

In addition, the indicator that can use any selected marker to transform as success.For example, can use " neomycin phosphotransferase " marker gene, perhaps use " hpt " marker gene to give resistance respectively to aminoglycoside antibiotics, kantlex and Totomycin.Other marker gene comprises " bar " marker gene, and it gives the resistance to the weedicide phosphinothricin; " DHFR " marker gene, it gives the resistance to methotrexate; And " ESPS " marker gene, it gives the resistance to the Round-up weedicide.Whether known in the prior art expression how to carry out this class marker gene is stably expressed in the genome of transformed plant cells with definite marker gene.Correspondingly, the technician how to know the trace labelling expression of gene with determine marker gene only in transformed plant cells the of short duration face of land reach.

In another aspect of the present invention, provide preparation stably to transform the method for plant, may further comprise the steps (1) and identify target gene; (2) separate relevant with described target gene leading or trail dna sequence dna; (3) can randomly modify described isolating leading or trail DNA; (4) operationally with described leading or trail DNA and be connected on the natural regulatory element to form expression cassette; (5) described expression cassette is inserted the P-DNA that is arranged on two carriers, wherein this pair carrier also carries an exercisable cell fission plain gene, feasible unexpected insertion of detecting by the phytokinin expression of gene of ectogenic other pair carrier sequence; (6) the two carriers that will modify are introduced in the edaphic bacillus; (7) use the conversion of LifeSupport mediation that the n DNA of resetting stably is incorporated in the genome of vegetable cell; (8) regeneration contains the vegetable cell of the n DNA of rearrangement; (9) discard and show and excessively to produce phytokinin phenotype and plant that can not holomorphosis; And (10) are kept with the indistinguishable expectation of unconverted plant plant and are used for further analysis.

The method of the trait expression of modifying selected plant species is provided in another aspect of the present invention.In one embodiment, this method comprises that (1) evaluation is with adorned proterties; (2) make up basically by separate from or the recombinant DNA molecules that constitutes of the natural genetic elements that belongs to selected plant species, wherein when the dna molecular of reorganization is integrated in the genome of selected plant species, modified the trait expression of conversion plant species; (3) use the conversion of LifeSupport mediation that the dna molecular of recombinating stably is incorporated in the cell of selected plant species; And (4) identify the adorned transformed plant of expression that shows proterties.

In a preferred embodiment, insert in the genome of expectation plant by the natural polynucleotide that will expect plant with two kinds of different edaphic bacilluss (Agrobacterium) strain infection explant.First edaphic bacillus bacterial strain can be transferred to n DNA the vegetable cell from the P-DNA carrier; The T-DNA that second bacterial strain can will have selected marker's expression cassette transfers in the vegetable cell.The example of a kind of carrier in back comprises what is called described herein " LifeSupport " carrier.By preferred can transient expression marker gene 1-10 days, 3-7 days, or 4-5 days plant, and subsequently explant is transferred in the regeneration culture medium, obtain numerous incidents, wherein the representative of part incident contains the plant of at least one polynucleotide copies, but lacks any copy of T-DNA or marker gene.

In another embodiment, use single edaphic bacillus bacterial strain, it has P-DNA carrier and LifeSupport carrier, wherein the P-DNA carrier has the natural interest genes or the polynucleotide of expectation between P-DNA edge sample sequence, and the LifeSupport carrier contains marker gene.Marker gene can, perhaps cannot be, or insert between other T-DNA sample edge sequence in P-DNA edge sample sequence, T-DNA edge sequence.

Therefore, in another preferred embodiment, the P-DNA carrier contains at least two expression cassettes, and one of them comprises natural screen or the selectable marker gene that is driven by natural promoter, is natural terminator then.

Expressed at least 2 days by preferred selection, and more preferably at least 5 days natural marker gene is expressed, subsequently outer plant is transferred in the regeneration culture medium, obtained numerous incidents, the DNA that its representative contains the introducing of at least one copy is incorporated into the plant in their genome with being stabilized.In preferred embodiments, the marker gene of plant origin coding 5-enol pyruvoyl-3-phosphoric acid shikimic acid (5-enolpyruvul-3-phosphoshikimic acid) synthase or tryptophan decarboxylase mutant.In a more preferred embodiment, selected marker thing coding salt tolerance.In the most preferred embodiment, the salt tolerance gene has the nucleotide sequence that shows among the SEQ ID 35, and is used to select transformation event in potato.

In another embodiment, the characteristics of the trait expression of process modification are to express the increase that increases, expresses reduction or non-detectable expression.

In another aspect of the present invention, provide the plant by the preparation of following method, identify (1) will adorned proterties; (2) make up the recombinant DNA molecules constitute by isolating genetic elements from selected plant species basically, wherein when the dna molecular of reorganization is integrated in the genome of selected plant species, the expression of this proterties in its modification plant transformed kind; (3) conversion by the LifeSupport mediation stably is incorporated into recombinant DNA molecules in the cell of selected plant species; And (4) identify the transformed plant that shows through the modification trait expression.

In aspect further, provide the method for modifying trait expression in the selected plant species.This method comprises that (1) evaluation is with adorned proterties; (2) make up the recombinant DNA molecules constitute by following part basically: (a) isolating genetic elements from selected plant species, wherein when genetic elements was integrated in the genome of selected plant species, its modified the expression of this proterties in conversion plant species; (b) isolating selected marker from identical plant species; (3) conversion by the LifeSupport mediation stably is incorporated into recombinant DNA molecules in the cell of selected plant species; (4) detect optionally marker gene; And (5) identify the transformed plant that shows through the modification trait expression.

One other aspect in, provide to show the plant of trait expression through modifying.In one embodiment, recombinant DNA molecules stably is incorporated in the genome of this plant, wherein this dna molecular basically by from mutually of the same race or from the affine plant of this kind property isolating genetic elements constitute, wherein this recombinant DNA molecules is modified the expression of proterties.

In another aspect of the present invention, provide the isolating nucleotide sequence that is known as " plant-DNA " (" P-DNA ").In a preferred embodiment, this P-DNA itself lacks any gene or its part, and describe with terminal T-DNA " edge sample " sequence, the T-DNA marginal nucleus nucleotide sequence of it and any strong edaphic bacillus bacterial strain has at least 50%, at least 75%, at least 90% or at least 95% sequence identity, its support is efficiently transferred to vegetable cell with whole P-DNA from edaphic bacillus.

In a preferred embodiment, " edge sample " sequence promotes and is convenient to the integration of coupled polynucleotide.In another preferred embodiment, the length of the end sequence of the P-DNA of each modification is 5-100 base pair, 10-80 base pair, 15-75 base pair, 15-60 base pair, 15-50 base pair, 15-40 base pair, 15-30 base pair, 16-30 base pair, 20-30 base pair, 21-30 base pair, 22-30 base pair, 23-30 base pair, 24-30 base pair, 25-30 base pair or 26-30 base pair.More preferably, the length of edge sample sequence is 20 and 28 Nucleotide.

In preferred embodiments, the edge sequence on the P-DNA of the present invention left side and the right be separate from and/or the natural genome that belongs to adorned plant, and nucleotide sequence the edge sequence with the T-DNA in any known edaphic bacillus (Agrobacterium) source is not identical.Therefore, in one embodiment, P-DNA edge sequence may have 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more a plurality of edaphic bacillus kind that is different from, as the Nucleotide of agrobacterium tumefaciens or root of hair plant Zymomonas mobilis (Agrobacterium rhizogens) T-DNA edge sequence.Perhaps, in another embodiment, P-DNA of the present invention edge or edge sample sequence and edaphic bacillus kind have at least 95%, at least 90%, at least 80%, at least 75%, at least 70%, at least 60% or at least 50% sequence identity as the T-DNA edge sequence of agrobacterium tumefaciens or root of hair plant Zymomonas mobilis (Agrobacterium rhizogens).More preferably, the T-DNA edge sequence of the P-DNA edge sequence of natural phant and edaphic bacillus (Agrobacterium) has and is greater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or 60% nucleotide sequence homology.

In a preferred embodiment, can be from Plant Genome separation edge sample sequence, then modify or suddenly change to change efficient, nucleotide sequence can be incorporated in another nucleotide sequence by this efficient.In another embodiment, can in edge of the present invention sample sequence, add or mix other polynucleotide sequence.Therefore, in another embodiment, can modify the right hand edge of the left hand edge of P-DNA or P-DNA so that it has 5 '-and 3 '-multiple clone site or other restriction enzyme site.In further embodiment, can modify P-DNA edge sequence and follow the main chain DNA of carrier not to be integrated into possibility in the Plant Genome with raising.

In one even preferred embodiment, from any plant, separate P-DNAs by in polymerase chain reaction, using degenerated primer.One preferred embodiment in, P-DNA describes with the end of 25 base pairs from potato, this end has 80 and 88% identity respectively with conventional T-DNA edge sequence, has the nucleotide sequence that SEQ ID NO.1 shows.In another the most preferred embodiment, P-DNA derives from wheat, describes with the end of 25 base pairs, and this end has 72% and 92% identity respectively with the T-DNA edge of routine, contains the nucleotide sequence that shows among the SEQ ID NO.34.

Can modify such P-DNA so that it comprises other polynucleotide between edge sample sequence.In a preferred embodiment, through the P-DNA that modification 5 '-constitute by first edge sample sequence that promotes DNA to shift, promotor, second edge sample sequence operationally linking polynucleotide, the terminator of the expectation on the promotor and also promote DNA to shift basically on 3 '-direction.In other embodiments, one or several copy of leader sequence, tailer sequence or the gene of the polynucleotide of expectation representative on justice and/or antisense orientation.In a more preferred embodiment, the P-DNA of modification contains the expression cassette of sudden change PPO gene and converting enzyme inhibitor gene.

Therefore, in preferred embodiments, the polynucleotide of expectation comprise the justice and the antisense sequences of leader sequence.In a more preferred embodiment, leader sequence is relevant with the inherent gene of selected plant species cell.In a more preferred embodiment, leader sequence is relevant with the gene of selecting from the group of following gene formation: the PPO gene, the R1 gene, L or H type alpha-glucan phosphorylase gene, UDP glucose Transglucosylase gene, the HOS1 gene, the adenosylhomocysteine hydrolase gene, II class styracin-4-'-hydroxylase gene, cinnyl-CoA reductase gene, the cinnyl alcohol dehydrogenase gene, caffeoyl coenzyme A O-methyl transferase gene, the actin depolymerizing factor gene, the Nin88 gene, Lol p 5 genes, the allergen gene, the P450 '-hydroxylase gene, ADP-glucose pyrophosphorylase gene, the proline dehydrogenase gene, in-1,4-beta-glucanase gene, zeaxanthin epoxidase gene, and 1-Aminocyclopropane-1-carboxylate synthase gene.

In another preferred embodiment, the polynucleotide of expectation comprise the justice and the antisense sequences of tailer sequence.In preferred embodiments, tailer sequence is relevant with the gene of selecting from the group of following gene formation: the PPO gene, the R1 gene, L or H type alpha-glucan phosphorylase gene, UDP glucose Transglucosylase gene, the HOS1 gene, the adenosylhomocysteine hydrolase gene, II class styracin-4-'-hydroxylase gene, cinnyl-CoA reductase gene, the cinnyl alcohol dehydrogenase gene, caffeoyl coenzyme A O-methyl transferase gene, the actin depolymerizing factor gene, the Nin88 gene, Lol p5 gene, the allergen gene, the P450 '-hydroxylase gene, ADP-glucose pyrophosphorylase gene, the proline dehydrogenase gene, in-1,4-beta-glucanase gene, zeaxanthin epoxidase gene, and 1-Aminocyclopropane-1-carboxylate synthase gene.

In preferred embodiments, the polynucleotide of expectation, as gene isolation from and/or natural belonging to by plant transformed.In another preferred embodiment, the polynucleotide of modifying or suddenling change and expect.In one embodiment, the sudden change of isolating polynucleotide may make the polynucleotide of expectation and its not mutant have and be greater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or 60% alienation.

In the preferred embodiment of the invention, the promotor that is positioned at the expression cassette of P-DNA is a constitutive promoter.In a more preferred embodiment, constitutive promoter is the promotor of ubiquitin-3 gene of potato.In addition preferred embodiment in, constitutive promoter is the promotor of ubiquitin-7 gene of potato.

In another embodiment, the promotor that is positioned at the expression cassette of P-DNA is adjustable promotor.In a more preferred embodiment, adjustable promotor is to temperature sensitive.In addition preferred embodiment in, so adjustable startup ci21A promotor or C17 promotor, each all is to separate (Schneider etc., Plant Physiol.113:335-45,1997 from potato; Kirch etc., Plant Mol Biol 33:897-909,1997) come out.

In another embodiment, the mode promotor of regulating the expression cassette that is positioned at P-DNA temporarily.In preferred embodiments, promotor is rbcS promotor (Ueda etc., Plant Cell 1:217-27,1989).

In another embodiment, be positioned at the promotor of the expression cassette of P-DNA by any one adjusting in dormin, wound, methyl jasmonate or the gibberic acid.In further embodiment, this promotor is the promotor of the promotor of the promotor of the promotor that is selected from Rab 16A gene, alpha-amylase gene or pin2 gene.

In another embodiment, the promotor that is positioned at the expression cassette of P-DNA is tissue-specific promotor.In particularly preferred embodiments, this promotor is an isolating GBSS promotor from potato.

In one embodiment, the invention provides and in intestinal bacteria (E.coli) and edaphic bacillus, to duplicate, and contain the P-DNA carrier of the P-DNA of P-DNA or process modification.In preferred embodiments, this carrier also contains the expression casette of phytokinin so that the incident that can select anti-main chain to integrate in its main chain.

In another preferred embodiment, the nucleotide sequence of expectation further comprises spacer element.In a more preferred embodiment, spacer element is Ubi intron sequences or GBSS intervening sequence.

In another preferred embodiment, the nucleotide sequence of expectation comprises the natural gene of the sudden change of encoding function inactivating protein, if express in transgenic plant, it reduces these proteic whole activity.In a more preferred embodiment, the genes encoding of this sudden change shortage is in conjunction with the polyphenoloxidase of the functionally inactive of the structural domain of copper.

In another preferred institute embodiment, the nucleotide sequence of expectation comprises activated proteic natural gene on the encoding function.In a more preferred embodiment, this genes encoding and tobacco vacuolus converzyme inhibitor homologous albumen.

In another embodiment, the terminator that is positioned at the expression cassette of P-DNA is 3 '-non-translational region of Ubi3 terminator sequence or selected plant species gene.

In another aspect of the present invention, provide a kind of method of modifying the target vegetable cell.In one embodiment, this method comprises: (1) uses the conversion of LifeSupport mediation that the P-DNA that modifies is inserted in the genome of at least one cell in the target vegetable cell; (2) observe the change whether phenotype is arranged in the target vegetable cell; Wherein the non-translated sequence of justice that promoter transcription is relevant with natural gene in the P-DNA that modifies and/or antisense to be reducing the expression of this natural gene, thereby modification target vegetable cell.In another preferred embodiment, a kind of gene of promoter transcription makes this gene of overexpression in the target vegetable cell in the P-DNA that modifies.

In yet another aspect, provide a kind of preparation to contain the method for the transgenic plant cells of the selected plant species of modifying P-DNA.This method comprises the vegetable cell with P-DNA carrier and the selected plant species of LifeSupport carrier cotransfection, wherein the LifeSupport carrier comprise both sides be the left hand edge of T-DNA and T-DNA right hand edge marker gene and be inserted into mutant virD2 gene in the carrier main chain, select the vegetable cell of presentation markup gene momently, and separate and to contain the modification P-DNA that is incorporated in its genome but do not contain vegetable cell from any Nucleotide of LifeSupport carrier.In preferred embodiments, this marker gene is given the resistance of anti-kantlex.It in the most preferred embodiment yeast ADH terminator.

In preferred embodiments, as the vegetable cell of the selected plant species that transforms target in culture.In another preferred embodiment, as the vegetable cell of the selected plant species that transforms target in plant.

The present invention also provides the plant of being chosen seeds, and it comprises the cell that at least one its genome contains the P-DNA of modification.In preferred embodiments, the P-DNA of this modification 5 '-on 3 '-direction basically by its function resemble T-DNA edge sequence and with heel with first end, the promotor of P-DNA sequence, be operably connected to the nucleotide sequence of the expectation on promotor and the terminator and constitute with the other P-DNA sequence that second end is described.In another embodiment, one or several copy of leader sequence, tailer sequence and the gene of the polynucleotide of expectation representative on justice and/or antisense orientation.

In another embodiment, imagination comprises the plant of cell that at least one its genome contains the P-DNA of modification.

In another aspect of the present invention, provide the method that reduces the genetic expression in the selected plant species.This method comprises with the P-DNA carrier carries out the conversion of LifeSupport mediation to the vegetable cell from selected plant species, and wherein the P-DNA with the modification of this carrier stably is incorporated in the genome of described vegetable cell.In another aspect of the present invention, what the P-DNA of modification comprised expectation can reduce the polynucleotide of expressing from the native gene of selected plant species.

In another aspect of the present invention, natural gene that can the selected plant species of mutagenesis also uses method of the present invention to be reintroduced in the plant.Preferably, use the P-DNA carrier with mutator gene, for example Tu Bian PPO gene integration is in the genome of vegetable cell.

The present invention also provides the method for not wishing the expression wanted that reduces polyphenol oxidase genes in the selected plant species.In preferred embodiments, this method comprises that the P-DNA that will modify is incorporated in the genome of selected plant species, the P-DNA of this modification only by from selected plant species or from the affine plant of selected plant species isolating nucleotide sequence constitute, 5 '-resemble T-DNA edge sequence by function basically on 3 '-direction and with heel first P-DNA end with flank P-DNA sequence; Promotor; The Nucleotide of expectation, it is the nucleotide sequence of trailing with the just direction of specific PPO gene-correlation; Specific PPO gene is trailed the antisense orientation sequence of nucleotide sequence; Terminator sequence, and constitute with the other P-DNA sequence that its function resembles the T-DNA edge of describing of second end, wherein promotor produces the double stranded rna molecule that can reduce specific PPO genetic expression, so reduces the blackspot scar in the specific tissue of plant.In another embodiment, from prior to the justice that obtains leading nucleotide sequence 5 '-non-translational region of specific PPO gene and the nucleotide sequence of antisense orientation.In further embodiment, can pass through another polynucleotide sequence, be called intron or " transcribed spacer " here, will separate with the justice of PPO gene-correlation and the leading or tailer sequence of antisense orientation.In preferred embodiments, leading or tailer sequence and potato PPO gene-correlation.In a more preferred embodiment, the leading or tailer sequence and the potato PPO gene-correlation of in potato tuber, expressing.In the most preferred embodiment, leading or tailer sequence and the potato PPO gene-correlation of in the stem tuber of potato, all expressing in all parts except epidermis.

The present invention also be provided at reduce acrylamide production in the stem tuber of selected plant species, duration of storage induces germination, phosphoric acid salt to accumulate and/or the cold method that sweetens of inducing.

In preferred embodiments, this method comprises the conversion of selected plant species being carried out the LifeSupport mediation with the P-DNA that modifies, the P-DNA of this modification only comprise from selected plant species or from the affine plant of selected plant species isolating nucleotide sequence, 5 '-on 3 '-direction basically by first P-DNA with left hand edge sample sequence, promotor, the nucleotide sequence of expectation, its be from the nucleotide sequence of the just direction of the leader sequence of R1 gene-correlation, the antisense orientation sequence of this leader sequence, terminator sequence, and right hand edge sample sequence constitutes.In case express, generation can reduce the leading-RNA duplex of R1 genetic expression, thereby the cold inductive of minimizing sweetens in plant.In another embodiment, the tailer sequence of representative of the nucleotide sequence of the justice of expectation and antisense orientation and R1 gene-correlation.In further embodiment, can pass through another polynucleotide sequence, be called intron or " transcribed spacer " here, the justice that will be relevant with R1 and the leader sequence or the tailer sequence of antisense orientation separate.

In another preferred embodiment, this method comprises the conversion of selected plant species being carried out the LifeSupport mediation with the P-DNA that modifies, wherein the P-DNA that should modify is similar to above-described P-DNA, but contains the leading or tailer sequence relevant with the alpha-glucan phosphorylase gene.

In another preferred embodiment, this method comprises with the P-DNA that modifies carries out the conversion that LifeSupport mediates to selected plant species, and wherein the P-DNA of this modification contains the converting enzyme inhibitor gene.

In another preferred embodiment, the P-DNA that uses the modification of describing in the paragraph in front accumulates with the extra undesired product that reduces Maillard reaction, and it occurs in and is rich in the food that the carbon aquation contains thing, between the heating period as potato tuber.These undesired products comprise relevant with various pathology always advanced glycation end products (AGEs).

The present invention also provides the method that improves Resistant starch level among plant and the food crop storage organ.

In preferred embodiments, this method comprises with the P-DNA that modifies carries out the conversion that LifeSupport mediates to selected plant species, and wherein the P-DNA of this modification contains the expression cassette of the fusion of the tailer sequence relevant with the II gene with Q-enzyme I.

The present invention also provides the isolating nucleotide sequence of the promotor that comprises main potato gbss gene of expressing and potato proteinase inhibitor gene in stem tuber.Isolating promotor has shown nucleotide sequence among SEQ ID NO.:6 and the SEQ ID NO.:40 respectively.

In one aspect, the invention provides the method for modifying selected plant trait, comprising:

A. the polynucleotide with expectation stably transform the cell of selected plant, and wherein Qi Wang polynucleotide constitute by selected plant, kindred plant or with the natural acid sequence of the interfertile plant of selected vegetalitas basically,

B. obtain the plant of stable conversion from the plant transformed cell, wherein plant transformed contains the polynucleotide of the expectation of stable integration in the genome, and the polynucleotide of wherein expectation are modified this proterties.

In preferred embodiment, this method further comprises the selected marker's cotransfection vegetable cell that is used in transient expression in the vegetable cell, identify the plant transformed cell, from the plant transformed cell, obtain plant transformed, wherein the selected marker is not stably integrated, and the polynucleotide of expectation stably are incorporated in the genome.

In preferred embodiments, the polynucleotide of expectation comprise the PPO gene, converting enzyme inhibitor gene, salt tolerance gene of P-DNA, GBSS promotor, Ubi7 promotor, Ubi3 promotor, PIP promotor, modification, the leader sequence relevant with R1, with Starch phosphorylase relevant leader sequence, with R1 relevant tailer sequence, tailer sequence, Ubi intron, GBSS transcribed spacer, the UbiT relevant with SBE.

In another preferred embodiment, " plant " of the present invention is monocotyledons, selects from the group that is made of wheat, turf, turfgrass, cereal grass, corn, paddy rice, oat, wheat, barley, jowar, orchid, Herba Iridis japonicae, lily, onion, banana, sugarcane, jowar and palm.

In another embodiment, " plant " of the present invention is dicotyledons, selects from the group that is made of avacado, potato, tobacco, tomato, beet, asparagus broccoli, cassava, sweet potato, pepper, cotton, poinsettia, leguminous plants, clover, soybean, Radix Dauci Sativae, strawberry, lettuce, Oak Tree, maple, English walnut, rose, peppermint, pumpkin, daisy and Root and stem of Cholla.

In another embodiment, transform the plant and the vegetable cell of the inventive method by agrobacterium-mediated being converted.Preferably, agrobacterium-mediated conversion depends at least a pair of carrier of use.In another embodiment, agrobacterium-mediated method for transformation uses first pair of carrier and second pair of carrier.First pair carrier contains the polynucleotide of expectation in preferred embodiments, and second pair of carrier contains marker gene selectively, and wherein optionally marker gene is operably connected on promotor and the terminator.

The method according to this invention, adorned proterties are to select from the group that following proterties constitutes: the health of raising and nutritive property, the storage that improves, the output that improves, the enhanced salt tolerance, enhanced heavy metal tolerance, the drought tolerance that improves, the disease tolerance that improves, the insect tolerance that improves, the water stress tolerance that improves, enhanced cold-peace frost tolerance, increase color, increase sweet taste, the raising vigor, improve taste, improve quality, reduce phosphate content, improve percentage of germination, improving little nutrition takes in, improving starch forms, improve the life-span of flower.

The present invention also comprises the plant by the inventive method preparation.

In yet another aspect, the invention provides the method for modifying selected plant trait, comprising:

(a) identify the proterties that to modify;

(b) make up first polynucleotide, its basically by from selected plant, kindred plant or with the interfertile plant of selected vegetalitas isolating natural genetic elements constitute, wherein natural genetic elements can be modified the expression of gene of this proterties of control;

(c) make up second polynucleotide that comprise the selected marker who is operably connected on promotor and the terminator;

(d) with the vegetable cell of the first and second polynucleotide cotransfections from selected plant;

(e) selection selected marker's transient expression;

(f) do not screen still not containing the vegetable cell that is incorporated into second dna molecular in the genome with first polynucleotide stable conversion; With

(g) plant of acquisition stable conversion from the plant transformed cell that the expression process that shows this proterties is modified.

In one embodiment, genetic elements comprises at least one in promotor, interest sequence, terminator, enhanser, intron, transcribed spacer or the regulatory element.In another embodiment, the method for claim 4 is wherein before second polynucleotide transfection, with first polynucleotide transfection of plant cells, perhaps conversely.

In one embodiment, the interest sequence is a gene.In another embodiment, this gene is sudden change or the R1 gene polyphenol oxidase genes of wild-type or sudden change or wild-type.In other embodiments, the interest sequence is leader sequence or tailer sequence, and wherein leading or tailer sequence is represented the upstream or the downstream sequence of vegetable cell natural gene.In another embodiment, the interest sequence comprises the leader sequence that is operably connected to the just direction on the antisense leader sequence.In another embodiment, the interest sequence comprises the tailer sequence that is operably connected to the just direction on the antisense tailer sequence.In another embodiment, promotor is an inducible promoter.In another embodiment, terminator is a yeast ADH terminator sequence.

According to the present invention, the leader sequence construction 5 '-on 3 '-direction, comprise the leader sequence on promotor, the just direction, the antisense sequences and the terminator of leader sequence, wherein the expression of leader sequence construction produces the double stranded rna molecule of being convenient to regulate relative genetic expression downwards.In other embodiments, the coding region of leader sequence and PPO gene, R1 gene, L type phosphorylase gene or alpha-glucan phosphorylase gene is relevant and be positioned at its upstream.

In another embodiment, trail construction 5 '-on 3 '-direction, comprise the tailer sequence on promotor, the just direction, the antisense sequences and the terminator of tailer sequence, wherein the expression of tailer sequence construction produces the double stranded rna molecule of being convenient to regulate the genetic expression of getting in touch with it downwards.In preferred embodiments, the coding region of tailer sequence and PPO gene, R1 gene, L type phosphorylase gene or alpha-glucan phosphorylase gene is relevant and be positioned at its downstream.

This method further comprises vegetable cell is exposed to second carrier that comprises identification element, and wherein this is marked at that the of short duration face of land reaches in the plant transformed, and is not incorporated in the genome that transforms plant with not being stabilized.In one embodiment, this mark is resistant gene, antibiotics resistance gene or the NPTII of weedicide.

Preferably, by the agrobacterium-mediated transformed plant cells that is converted.In one embodiment, agrobacterium-mediated conversion depends at least one two carrier of use.In another embodiment, the method for transformation of this edaphic bacillus (Agrobacterium) mediation uses first pair of carrier and second pair of carrier.In other embodiments, first pair of carrier has first polynucleotide, and second pair of carrier has second polynucleotide.

The invention provides the method that another modifies genetic expression in the selected plant, comprising:

(a) identify functional gene;

(b) make up basically by from selected plant, with selected plant plant of the same race or with the interfertile plant of selected vegetalitas first polynucleotide that constitute of isolating natural genetic elements, wherein this natural genetic elements can be modified this expression of gene;

(c) make up second polynucleotide that comprise functionally selected property marker gene;

(e) selection selected marker's transient expression;

(f) screening stably transforms with first polynucleotide, but does not contain the vegetable cell that is incorporated into second polynucleotide in the genome; With

(g) from showing that this expression of gene is through obtaining to transform plant the transformed plant cells of modifying.

Preferably, by the agrobacterium-mediated transformed plant cells that is converted.In one embodiment, agrobacterium-mediated conversion depends at least one two carrier of use.In another embodiment, agrobacterium-mediated method for transformation uses first pair of carrier and second pair of carrier.In other embodiments, first pair of carrier has first polynucleotide, and second pair of carrier has second polynucleotide.

In another embodiment, first polynucleotide comprise the PPO gene, converting enzyme inhibitor gene, salt tolerance gene of P-DNA, GBSS promotor, Ubi7 promotor, Ubi3 promotor, PIP promotor, modification, the leader sequence relevant with R1, with Starch phosphorylase relevant leader sequence, with R1 relevant tailer sequence, with SBE among relevant tailer sequence, Ubi intron, GBSS transcribed spacer, the UbiT at least one.

In another embodiment, second polynucleotide comprise at least one in virD2 polynucleotide, codA polynucleotide and the codA ∷ upp fusion polynucleotides of selected marker, Ω-sudden change.

The present invention also comprises the plant of preparation by this method.

In other embodiments, the transgenic plant of expression that provide demonstration to compare proterties with the non-transgenic plant in its source through modifying, wherein the polynucleotide with the expectation that is made of natural genetic elements basically stably transform transgenic plant, this natural genetic elements from this plant, kindred plant or with the interfertile plant of this vegetalitas separate, wherein these polynucleotide are modified the expression of proterties.

In another embodiment, proterties is selected from the group that following proterties constitutes: the health of raising and nutritive property, the storage that increases, the output that improves, the enhanced salt tolerance, enhanced heavy metal tolerance, the drought tolerance that improves, the disease tolerance that improves, the insect tolerance that improves, the water stress tolerance that improves, enhanced cold-peace frost tolerance, the color that increases, the sweet taste that increases, the vigor that improves, the taste that improves, the structure of improving, the phosphate content that reduces, the percentage of germination that improves, the little nutrition that improves is taken in, the starch that improves is formed, the life-span of the flower that improves.

In another embodiment, the polynucleotide of expectation comprise the PPO gene, converting enzyme inhibitor gene, salt tolerance gene of P-DNA, GBSS promotor, Ubi7 promotor, Ubi3 promotor, PIP promotor, modification, the leader sequence relevant with R1, with Starch phosphorylase relevant leader sequence, with R1 relevant tailer sequence, with SBE among relevant tailer sequence, Ubi intron, GBSS transcribed spacer, the UbiT at least one.

The present invention also comprises size at the isolating edge sample nucleotide sequence from 20 to 100bp scopes, and it has 52% and 96% sequence identical with the T-DNA edge sequence of agrobacterium tumefaciens.In preferred embodiments, isolating nucleotide sequence is isolating from monocotyledons, and monocotyledons is selected from the group that is made of wheat, turf, turfgrass, cereal grass, corn, paddy rice, oat, wheat, barley, jowar, orchid, Herba Iridis japonicae, lily, onion, banana, sugarcane, jowar and palm.In another embodiment, nucleotide sequence is isolating from dicotyledons, and this dicotyledons is selected from the group that is made of potato, tobacco, tomato, beet, asparagus broccoli, cassava, sweet potato, pepper, cotton, poinsettia, leguminous plants, clover, soybean, Radix Dauci Sativae, strawberry, lettuce, Oak Tree, maple, English walnut, rose, peppermint, pumpkin, daisy and Root and stem of Cholla.

In another embodiment, isolating nucleotide sequence is isolating from potato, and has the nucleotide sequence that shows in SEQ ID NO.94 or 95.In preferred embodiments, isolating nucleotide sequence and the T-DNA edge sequence of agrobacterium tumefaciens have 52% sequence identical.The present invention includes the carrier that comprises such nucleotide sequence.

The present invention also provides the method for preparation with the plant of the polynucleotide stable conversion of expectation, comprising:

(a) separating both sides from this plant is P-DNA of edge sample sequence, and wherein the T-DNA edge sequence of edge sample sequence and agrobacterium tumefaciens has 52% to 96% sequence identity;

(b) between P-DNA edge sample sequence, insert the polynucleotide of expectation to form the P-DNA construction; With

(c) be converted from the vegetable cell of this plant with the P-DNA construction; With

(d) from transformed plant cells, regain plant with P-DNA construction stable conversion.

In one embodiment, on the carrier that constitutes by the main chain integration marker gene, carry this P-DNA construction, and select not contain the plant transformed cell of main chain integration marker gene.In another embodiment, this main chain integration marker gene is the cell fission plain gene.In another embodiment, do not select to show the plants shoots of excessive production phytokinin phenotype.In another embodiment, the main chain integration marker gene is the IPT gene, does not select the display abnormality phenotype or can not produce the plants shoots of root.

In other embodiments, vegetable cell is from monocotyledons, and this monocotyledons is selected from the group that is made of wheat, turf, turfgrass, cereal grass, corn, paddy rice, oat, wheat, barley, jowar, orchid, Herba Iridis japonicae, lily, onion, banana, sugarcane, jowar and palm.

In another embodiment, this vegetable cell is from dicotyledons, and this dicotyledons is selected from the group that is made of potato, tobacco, tomato, beet, asparagus broccoli, cassava, sweet potato, pepper, cotton, poinsettia, leguminous plants, clover, soybean, Radix Dauci Sativae, strawberry, lettuce, Oak Tree, maple, English walnut, rose, peppermint, pumpkin, daisy and Root and stem of Cholla.

Preferably, by the agrobacterium-mediated transformed plant cells that is converted.In one embodiment, agrobacterium-mediated conversion depends at least one two carrier of use.In another embodiment, this agrobacterium-mediated method for transformation uses first pair of carrier and second pair of carrier.In other embodiments, first pair of carrier has first polynucleotide, and second pair of carrier has second polynucleotide.In a further embodiment, the second pair of carrier comprises at least one in the virD2 gene of negative selected marker and Ω-sudden change, wherein Yin Xing selected marker is positioned at T-DNA right hand edge and T-DNA left hand edge, and the virD2 gene of wherein Ω-sudden change is positioned at the main chain of second pair of carrier.In preferred embodiments, second pair of carrier comprises the negative selected marker who is positioned at T-DNA right hand edge and T-DNA left hand edge, and the virD2 gene that is positioned at second pair of carrier master intrachain Ω-sudden change.

The present invention also is provided at 5 '-P-DNA that constitutes by a T-DNA edge sample sequence, promotor, the polynucleotide sequence that is operably connected to the expectation on the promotor, terminator and the 2nd T-DNA edge sample sequence basically on 3 '-direction, and wherein edge sample sequence and T-DNA edge sequence have the sequence identity less than 100%.

In preferred embodiments, the polynucleotide of T-DNA edge sample sequence, promotor, expectation and terminator are all separated from identical plant, identical plant species or the interfertile plant of property.

In another embodiment, P-DNA and then constitute by the selected marker basically.

In another embodiment, polynucleotide, terminator and the selected marker of T-DNA edge sample sequence, promotor, expectation separate from identical plant, identical plant species or the interfertile plant of property.

In another embodiment, the polynucleotide sequence of the expectation among the P-DNA is the upstream or the downstream sequence of gene coding region, and wherein this upstream sequence is a leader sequence, and wherein downstream sequence is a tailer sequence.In this embodiment, T-DNA edge sample sequence, promotor, leader sequence, tailer sequence, terminator and selected marker separate from identical plant, identical plant species or the interfertile plant of property.

In another embodiment, by the invention provides the carrier that comprises such P-DNA construction.

In another embodiment, promotor is adjustable promotor.In another embodiment, adjustable promotor is to temperature sensitive.In preferred embodiments, adjustable promotor is a wheat wcs120 promotor.In another embodiment, promotor is under the sequential regulation and control.In another embodiment, promotor is the carboxylase promotor.In further embodiment, the carboxylase promotor is a corn carboxylase promotor.

Can be by any promotor of regulating in dormin, wound, methyl jasmonate or the gibberic acid.In another embodiment, promotor is the promotor that is selected from Rab 16A gene promoter, αDian Fenmei gene promoter or pin2 gene promoter.In another embodiment, promotor is tissue-specific promotor.

In other embodiments, leader sequence is the part of 5 '-non-translational region of the native gene of selected plant species cell.In another embodiment, 5 '-non-translational region is positioned at the upstream of the initiator codon of gene, wherein this gene is from by the PPO gene, the R1 gene, the HOS1 gene, the adenosylhomocysteine hydrolase gene, II class styracin-4-'-hydroxylase gene, cinnyl-CoA reductase gene, the cinnyl alcohol dehydrogenase gene, caffeoyl coenzyme A O-methyl transferase gene, the actin depolymerizing factor gene, the Nin88 gene, Lol p 5 genes, the allergen gene, the P450 '-hydroxylase gene, ADP-glucose pyrophosphorylase gene, the proline dehydrogenase gene, in-1,4-beta-glucanase gene, zeaxanthin epoxidase gene, and select in the group of 1-Aminocyclopropane-1-carboxylate synthase gene formation.

In another embodiment, tailer sequence is the part of 3 '-non-translational region that is positioned at the gene in the terminator codon downstream that is selected from following gene: the PPO gene, the R1 gene, the HOS1 gene, the adenosylhomocysteine hydrolase gene, II class styracin-4-'-hydroxylase gene, cinnyl-CoA reductase gene, the cinnyl alcohol dehydrogenase gene, caffeoyl coenzyme A O-methyl transferase gene, the actin depolymerizing factor gene, the Nin88 gene, Lol p5 gene, the allergen gene, the P450 '-hydroxylase gene, ADP-glucose pyrophosphorylase gene, the proline dehydrogenase gene, in-1,4-beta-glucanase gene, zeaxanthin epoxidase gene, and 1-Aminocyclopropane-1-carboxylate synthase gene.

Carrier of the present invention may further include spacer element, and this spacer element is Ubi intron sequences or GBSS transcribed spacer sequence.In another embodiment, carrier comprises terminator, and this ends at is 3 '-non-translational region of Ubi3 terminator sequence or endogenous plant gene.

In another embodiment, carrier comprises optionally being connected to the selected marker on the constitutive promoter and being operably connected to slander leads Cre gene on the type promotor, and wherein the both sides of selected marker and Cre gene are the recognition site of first recombinase and the recognition site of second recombinase.In another embodiment, the recognition site of the recognition site of first recombinase and second recombinase is the lox site.

In another embodiment, so induction type starts the promotor or the sequential promotor of temperature sensitive promotor, chemical induction.In another embodiment, inducible promoter is Ha hsp 17.7 G4 promotors, wheat wcs120 promotor, Rab 16A gene promoter, αDian Fenmei gene promoter, pin2 gene promoter, carboxylase promotor.In another preferred embodiment, the marker gene that further comprises plant origin.In another preferred embodiment, the marker gene of plant origin is enol pyruvoyl-3-phosphoric acid shikimic acid synthase gene.

In another aspect of the present invention, the method of modified plant cell is provided, comprise the P-DNA sequence is incorporated in the genome of vegetable cell, wherein this P-DNA 5 '-on 3 '-direction basically by a T-DNA edge sample sequence, promotor, be operably connected to the polynucleotide sequence of the expectation on the promotor, terminator and the 2nd T-DNA edge sample sequence constitute, wherein edge sample sequence and T-DNA edge sequence have the sequence identity less than 100%, T-DNA edge sample sequence wherein, promotor, the polynucleotide sequence of expectation all separates certainly or the natural genome that belongs to vegetable cell with terminator, wherein Qi Wang polynucleotide comprise the leader sequence relevant with the upstream of plant gene or downstream non-coding region or the justice and the antisense sequences of tailer sequence, wherein the expression of Qi Wang polynucleotide produces the double-stranded RNA transcript of the target gene relevant with the polynucleotide of expectation, thus the modified plant cell.

The present invention also comprises the method for modified plant, comprising:

(i) with at least one cell in the carrier transfection plant of the present invention;

(ii) select to express the cell of functional selected marker;

(iii) separate the cell of expressing functional selected marker;

The (iii) expression of Cre gene in isolated cells of inducing function;

(iV) cell of culture of isolated; With

(ii) observe the phenotype of cultured cells;

Wherein different with the vegetable cell of untransfected phenotypes shows that the target vegetable cell is modified.

In preferred embodiments, the selection step of this method and other method of the present invention is tested and appraised the antibiotic cell of opposing and carries out.

In yet another aspect, identify that its genome contains the method for the target vegetable cell of P-DNA, comprise with carrier of the present invention and the carrier cotransfection plant target cell that derives from second edaphic bacillus, it is the marker gene of T-DNA left hand edge and T-DNA right hand edge and the virD2 gene of Ω-sudden change that this second carrier comprises both sides, wherein P-DNA is incorporated in the genome of plant target cell, the any part that does not wherein have to derive from the carrier of second edaphic bacillus is incorporated in the genome of plant target cell, in preferred embodiments, the mark in the carrier that derives from second edaphic bacillus is a neomycin phosphotransferase gene.

In yet another aspect, provide and identify that its genome contains the method for the target vegetable cell of at least a portion of integrating box, and then comprise the cell that is chosen in temporary transient growth survival in the substratum that contains kantlex, wherein the genome of selected cell only contains the integration box.In one embodiment, described target vegetable cell is in plant.The present invention also comprises and comprises the plant that at least one its genome contains the cell of such P-DNA.

The present invention comprises also and comprises at least one its genome by the plant of manual operation with the cell of the nucleic acid that only contains plant origin that wherein the cell of this plant does not contain the exogenous nucleic acid that is incorporated in the cellular genome.

The present invention also comprises the polynucleotide of the polynucleotide sequence that comprises SEQ ID NO.93, and wherein the length of polynucleotide is 20 to 80 Nucleotide.In one embodiment, the length of polynucleotide is 21 to 70 Nucleotide, 22 to 50 Nucleotide, and 23 to 40 Nucleotide perhaps are 24 to 30 Nucleotide.

In yet another aspect, present invention resides in stem tuber specificity promoter shown among the SEQ ID NO.40.

The present invention also comprises the method that does not contain the transgenic plant cells that stably is incorporated into the selected marker among the nuclear DNA based on the preparation of edaphic bacillus, comprising:

A. make up the first pair of carrier that is made of polynucleotide, these polynucleotide are operably connected to the functional gene formation of the expectation on T-DNA edge or the T-DNA edge sample sequence basically by 5 ' and 3 ' end at the functional gene of expecting;

B. make up by 5 ' and 3 ' end and be operably connected to second pair of carrier that the functionally selected marker gene on T-DNA edge or the T-DNA edge sample sequence constitutes at functional selectable marker gene;

C. with the following bacterial strain cell that cultivates plants:

I. the edaphic bacillus bacterial strain that has first and second pairs of carriers; Or

Ii. the first edaphic bacillus bacterial strain and the second edaphic bacillus bacterial strain that has second pair of carrier that have first pair of carrier;

D. select wherein the functional gene of expectation is incorporated among the plant nuclear DNA, and not have marker gene optionally to be incorporated into vegetable cell among the nuclear DNA of plant, follow containing on the substratum of suitable selective agent and cultivate reasonable time.

In preferred embodiments, the selected marker is herbicide resistance gene or antibiotics resistance gene.In another preferred embodiment, antibiotics resistance gene is the nNPTII gene.In another embodiment, antibiotics resistance gene is the nptII structure gene that is operably connected on the terminator of the promotor of ubiquitin-7 gene and Alcohol Dehydrogenase from Yeast 1 (ADH) gene.According to this method,, then cultivate, perhaps conversely with the second edaphic bacillus bacterial strain at first with the first edaphic bacillus bacterial strain culturing plants cell.

In preferred embodiments, first pair of carrier also comprises two integration marker genes, and this gene can be used for detecting the vegetable cell with two carrier main chain sequence stable conversion.In another embodiment, two vector integration marker gene are selected from the group that is made of herbicide resistance gene, antibiotics resistance gene or NPTII.In another embodiment, second pair of carrier so be included in bacterium and the gene of Isocytosine deaminase (codA) and uracil phosphoribosyl transferase (upp) between gene fusion, this gene fusion is inserted between T-DNA or T-DNA edge sample sequence, vegetable cell is exposed to 5-flurocytosine, then cultivates so that selects to resist those with second pair of carrier plant transformed cell with the first and second edaphic bacillus bacterial strains.

In another embodiment, the second pair of carrier also comprises the gene that reduces the possibility that main chain integrates.In one embodiment, such gene is the virD2 gene of Ω-sudden change, and wherein the virD2 gene of Ω-sudden change reduces the frequency among the nuclear DNA that the selected marker is incorporated into plant.

The present invention also comprises the isolating nucleotide sequence that comprises isolating GBSS promotor from potato.In preferred embodiments, this isolating nucleotide sequence has the nucleotide sequence of SEQ ID.NO.6 or 13.

Brief description of the drawings

Fig. 1. some P-DNA carriers of Shi Yonging illustrates in the present invention.The P-DNA district shows with grey box." ipt " equals ipt expression of gene box; " npt " equals nptII expression of gene box; The PPO expression of gene box that " mPPO " equals to modify; " INH " equals converting enzyme inhibitor expression of gene box; " GUS " equals the expression cassette of gus gene; " LPPO " equals the justice of the leader sequence relevant with the PPO gene and the expression cassette of antisense copy; " LPH " equals the justice of the leader sequence relevant with phosphorylase gene and the expression cassette of antisense copy; " Alf " equals the expression cassette of potato Alfin homologue.Detailed description is seen text.

Fig. 2. no expression of gene box.

Fig. 3. the proteic sequence contrast of potato and tobacco converting enzyme inhibitor." St " equals potato; " Nt " equals tobacco.

Fig. 4. with the contrast of the tailer sequence of various PPO gene-correlations.

Fig. 5. some LifeSupport carriers of Shi Yonging illustrates in the present invention." codA " is codA expression of gene box; " codA ∷ upp " is the codA expression of gene box that is fused on the upp; " Ω virD2 " is Ω virD2 expression of gene box.

Detailed description of the preferred embodiments

" precise breeding " of the present invention strategy improves economical character, nutritive value and the health characteristic of plant and farm crop, not in the nucleic acid introduced plant kind genome with the nucleic acid of the unknown or external source species, and do not produce undesired phenotype or deleterious side effect.

Therefore, the method that the invention provides transgenic plant and prepare such plant, this method are not incorporated into the nucleic acid of non-plant kind in the genome of this plant.Nucleic acid, promotor, regulatory element, other noncoding gene order, mark, polynucleotide and to be incorporated into gene in the genome of selected plant preferably all be from being separated by plant transformed, the plant mutually of the same race that will be transformed or with being transformed the interfertile plant of vegetalitas.According to method described herein, such " natural " nucleic acid can add in the expression cassette jointly by mutagenesis, modification or with other natural acid, and is incorporated into again in the genome of selected plant.Correspondingly, only use the nucleic acid of selected plant oneself, or the nucleic acid of use and the affine plant of selected vegetalitas changes genotype and the phenotype of transgenic plant.

For the ease of this production of transgenic plants, utilization of the present invention is not that all T-DNA carriers of using in agrobacterium-mediated conversion in fact all are integrated into the fact in the Plant Genome, that is although carrier may be taken in by vegetable cell, actual integration incident may not take place.According to the present invention, can use such carrier that the selected marker is taken in the vegetable cell.Expressed how long screen vegetable cell by measuring marker gene then, marked whether to be incorporated in the Plant Genome determining with being stabilized.Correspondingly, selected marker's vegetable cell is only expressed in expectation momently, because the selected marker has been taken in their representatives, but is not integrated into cell in their genome.

Therefore, by the plant of using such " labeled vector " and also containing the carrier cotransformation of the natural gene of expectation or polynucleotide with another, the vegetable cell that can select two carriers all to take in determines that from these cells which cell has the gene that only contains expectation or the genome of polynucleotide.Can modify " labeled vector " and then reduce marker and will be integrated into possibility in the Plant Genome.The invention provides this class " labeled vector " that exists with " LifeSupport " carrier format.

Unless otherwise defined, the implication of all technology used herein and scientific terminology is identical with the implication of those skilled in the art's common sense.Usually, the laboratory method of title used herein, cell cultures, molecular genetics, and nucleic acid chemistry described herein and hybridization all are those known and normally used methods in the prior art.Use that recombinant nucleic acid method, polynucleotide are synthetic, microorganism culturing, cell cultures, tissue culture, conversion, transfection, transduction, analytical chemistry, Synthetic Organic Chemistry, chemosynthesis, chemical analysis, and the medicine preparation and the standard technique of carrying.Usually, carry out enzymatic reaction and purifying and/or separating step according to manufacturer's explanation.Generally come technical application and method according to disclosed ordinary method, for example at Molecular cloning a laboratory manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and Current protocols in molecular biology, John Wiley﹠amp; Sons, Baltimore, MD (1989) disclosed method.

Aminoacid sequence: aminoacid sequence used herein comprises oligopeptides, peptide, polypeptide or albumen and fragment thereof, its separate from, belong to or naturally occurring in plant or synthetic preparation, but comprise the nucleotide sequence of endogenous counterpart.

Manned: " manned " used herein meaning is by method manual or by mechanical means or reorganization, as pass through gene engineering, move, arrangement, operation or controlling plant or vegetable cell so that produce compare with not operated naturally occurring counterpart have different biology, biological chemistry, morphology or physiology phenotype and/or genotypic plant or vegetable cell.

Vegetative propagation: by insert from leaf, stem is inserted, root is inserted, the method for generation whole plants is come the production filial generation the tuberogemma, stolon, single plant protoplast, corpus callosum etc., does not comprise the fusion of gamete.

Main chain: remove the T-DNA that is used to shift or the nucleotide sequence of the two carriers outside the P-DNA sequence.

Edge and edge sample sequence: " edge sequence " is the specific sequence that derives from edaphic bacillus.Typically, left hand edge sequence and right hand edge sequence are positioned at the both sides of T-DNA, and their boths are as the recognition site of the catalytic cleavage reaction of virD2.Such activity discharges the nucleic acid between such edge sequence.The example of the edge sequence in 2 that sees the following form.The nucleic acid that discharges and virD2 and virE2 formation complex body target often are incorporated into nucleic acid plant nucleolus in the vegetable cell genome.Usually, use two edge sequences, left hand edge and right hand edge are incorporated into the nucleotide sequence between them in another nucleotide sequence.Also may perhaps more than two edge sequences, finish the integration of expectation nucleic acid by this way only with an edge sequence.

According to the present invention, " edge sample " sequence is the selected plant species from modifying, perhaps with the affine plant of the plant species that will modify in isolating, its function resembles the edge sequence of edaphic bacillus (Agrobacterium).That is, edge of the present invention sample sequence promotes and is convenient to the integration of coupled polynucleotide.DNA of plants of the present invention, promptly P-DNA preferably contains edge sample sequence.

The edge sample sequence length of P-DNA is 5-100bp, 10-80bp, 15-75bp, 15-60bp, 15-50bp, 15-40bp, 15-30bp, 16-30bp, 20-30bp, 21-30bp, 22-30bp, 23-30bp, 24-30bp, 25-30bp, or 26-30bp.

Edge of the present invention sample sequence can be separated from any plant, separates as from potato and wheat.See SEQ ID NO.1 and SEQ ID NO.34, these sequences contain isolating edge sample sequence from potato and wheat respectively at arbitrary end.Therefore, the P-DNA left side of the present invention's use is to separate certainly and/or the natural genome that belongs to the plant that will modify with the right side edge sequence.P-DNA edge sample sequence lists at nucleotides sequence and is different from any known T-DNA edge sequence that derives from edaphic bacillus.Therefore, P-DNA edge sample sequence may have 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more a plurality of edaphic bacillus kind that is different from, as the Nucleotide of the T-DNA edge sequence of agrobacterium tumefaciens or root of hair plant Zymomonas mobilis.Promptly, P-DNA of the present invention edge sequence or edge sample sequence and edaphic bacillus kind, T-DNA edge sequence as agrobacterium tumefaciens or root of hair plant Zymomonas mobilis has at least 95%, at least 90%, at least 80%, at least 75%, at least 70%, at least 60% or at least 50% sequence identity, but be not 100% sequence identity.Descriptive term used herein " P-DNA edge " and " P-DNA edge sample " can exchange.

Natural P-DNA edge sequence lists T-DNA edge sequence with edaphic bacillus at nucleotides sequence to be had and is greater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 52%, 51% or 50% similarity.Thereby edge sample sequence can separate from Plant Genome, and modified or suddenly change and nucleotide sequence can be incorporated into efficient in another nucleotide sequence to change by it.Can in edge of the present invention sample sequence, add or mix other polynucleotide sequence.Therefore, can modify the right side edge sequence of the left side edge sequence of P-DNA or P-DNA so that it has 5 '-and 3 '-multiple clone site or other restriction enzyme site.The edge sequence that can modify P-DNA is incorporated into possibility in the Plant Genome to improve the main chain DNA that will not follow carrier.

Tabulate down and 2 described known T-DNA edge sequence and be accredited as the sequence of edge sample sequence here.The sequence that none is accredited as " edge sample " in table 2 was accredited as in the past has T-DNA edge spline structure.In polymerase chain reaction, use degenerated primer from potato gene group DNA, to separate the edge sample sequence of potato by method of the present invention.The present invention includes the polynucleotide that use any P-DNA edge sample sequence to connect altogether transfers in the genome of vegetable cell.

In fact, the present invention includes any edge sample sequence of nucleotide sequence structure with SEQ ID NO.93:ANGATNTATN6GT (SEQ IDNO.93), wherein " N " is any Nucleotide, as those Nucleotide of " A ", " G ", " C ", " T " representative.This sequence representative is by the consensus sequence of the edge sample nucleic acid of the present invention's evaluation.

Table 2. " edge " and " edge sample " sequence

Edaphic bacillus T-DNA edge sequence
Edaphic bacillus T-DNA edge sequence		TGACAGGATATATTGGCGGGTAAAC(SEQ ID NO.41)	Edaphic bacillus nopaline bacterial strain (RB)
TGGCAGGATATATTGTGGTGTAAAC(SEQ ID NO.42)	Edaphic bacillus nopaline bacterial strain (LB)	TGACAGGATATATTGGCGGGTAAAC(SEQ ID NO.41)	Edaphic bacillus nopaline bacterial strain (RB)
TGGCAGGATATATTGTGGTGTAAAC(SEQ ID NO.42)	Edaphic bacillus nopaline bacterial strain (LB)	TGGCAGGATATATACCGTTGTAATT(SEQ ID NO.43)	Edaphic bacillus octopine bacterial strain (RB)
CGGCAGGATATATTCAATTGTAATT(SEQ ID NO.44)	Edaphic bacillus octopine bacterial strain (LB)	TGGCAGGATATATACCGTTGTAATT(SEQ ID NO.43)	Edaphic bacillus octopine bacterial strain (RB)
CGGCAGGATATATTCAATTGTAATT(SEQ ID NO.44)	Edaphic bacillus octopine bacterial strain (LB)	TGGTAGGATATATACCGTTGTAATT(SEQ ID NO.45)	The LB mutant
TGGCAGGATATATGGTACTGTAATT(SEQ ID NO.46)	The LB mutant	TGGTAGGATATATACCGTTGTAATT(SEQ ID NO.45)	The LB mutant
TGGCAGGATATATGGTACTGTAATT(SEQ ID NO.46)	The LB mutant	YGRYAGGATATATWSNVBKGTAAWY(SEQ ID NO.47)	The edge motif
Edge sample sequence		YGRYAGGATATATWSNVBKGTAAWY(SEQ ID NO.47)	The edge motif
Edge sample sequence		CGGCAGGATATATCCTGATGTAAAT(SEQ ID NO.48)	Rhizobium leguminosarum
TGGCAGGAGTTATTCGAGGGTAAAC(SEQ ID NO.49)	The thermophilic anaerobic bacillus	CGGCAGGATATATCCTGATGTAAAT(SEQ ID NO.48)	Rhizobium leguminosarum
TGGCAGGAGTTATTCGAGGGTAAAC(SEQ ID NO.49)	The thermophilic anaerobic bacillus	TGACAGGATATATCGTGATGTCAAC(SEQ ID NO.50)	Arabidopis thaliana
GGGAAGTACATATTGGCGGGTAAAC(SEQ ID NO.51)	Arabidopis thaliana CHR1v07142002	TGACAGGATATATCGTGATGTCAAC(SEQ ID NO.50)	Arabidopis thaliana
GGGAAGTACATATTGGCGGGTAAAC(SEQ ID NO.51)	Arabidopis thaliana CHR1v07142002	TTACAGGATATATTAATATGTATGA(SEQ ID NO.52)	Paddy rice AC078894
TAACATGATATATTCCCTTGTAAAT(SEQ ID NO.53)	The people clones HQ0089	TTACAGGATATATTAATATGTATGA(SEQ ID NO.52)	Paddy rice AC078894
TAACATGATATATTCCCTTGTAAAT(SEQ ID NO.53)	The people clones HQ0089	TGACAGGATATATGGTAATGTAAAC(SEQ ID NO.54)	Potato (left side edge sequence) *
TGGCAGGATATATACCGATGTAAAC(SEQ ID NO.55)	Potato (right side edge sequence) *	TGACAGGATATATGGTAATGTAAAC(SEQ ID NO.54)	Potato (left side edge sequence) *

Y=C or T; R=A or G; K=G or T; M=A or C; W=A or T; S=C or G; V=A, C, or G; B=C, G, or T.

The registration number of edge sample sequence is: rice chromosome 10 BAC OSJNBa0096G08 genome sequences (AC078894.11); Arabidopsis thaliana chromosome 3 (NM_114337.1); Arabidopsis thaliana chromosome 1 (NM-105664.1); Thermophilic anaerobic bacillus strain MB4T, the 118th district (AE013091.1) in 244 districts of full gene group; The people clones HQ0089 (AF090888.1); Rhizobium clone: rhiz98e12.q1k. ^*Obtain and separated potato left side and right side edge sequence according to the method for the present invention of present description.

Carrier DNA: " carrier DNA " is to be used for carrying some genetic elements and they are delivered to the dna fragmentation of vegetable cell.In the foreign DNA of routine shifted, this carrier DNA usually was the T-DNA of edaphic bacillus, describes with the edge sequence.Here the carrier DNA of describing is to obtain from the selected plant species that will modify, and contain on the structure He on the function and be different from T-DNA edge sequence, but with the end that such T-DNAs has same ability, this ability can support DNA to transfer to vegetable cell or some other Eukaryotic nuclear and subsequently this DNA is incorporated in so Eukaryotic gene from edaphic bacillus.

Basically by ... constitute: be limited to the inclusion of those elements basically by the composition of some element " formation ", and those do not influence the fundamental characteristics of composition of the present invention and the element of new features in fact.Therefore, as long as said composition does not influence fundamental characteristics of the present invention and new features, promptly do not contain be not from selected plant species or with the affine plant of selected plant species isolating foreign DNA, then said composition can be considered to the component of the present composition, be characterized in term " basically by ... constitute " expression.

Degenerated primer: " degenerated primer " is the oligonucleotide that contains enough nucleotide diversities, and when to similar, but when not being strict homologous sequence hybridization, it can yield to base mispairing.

Dicotyledons (dicot); Its embryo has the flowering plant of two cotyledons.The example of dicotyledons includes but not limited to tobacco, tomato, potato, sweet potato, cassava, the leguminous plants that comprises clover and soybean, Radix Dauci Sativae, strawberry, lettuce, Oak Tree, maple, English walnut, rose, peppermint, pumpkin, daisy and Root and stem of Cholla.

Regulate sequence; Refer to it is standard and known those sequences for a person skilled in the art, it can be contained in the expression vector, to improve in botanical system and/or to make transcribing maximization or improving and/or make the RNA translation maximization that obtains of interest genes.These include but not limited to promotor, peptide output signal sequence, intron, polyadenylation and Transcription Termination site.The modification of nucleic acids construction also is knownly (to see Rogers etc. for example, 260 J.Biol.Chem.3731-38,1985 with the method that improves the expression level in plant in the prior art usually; Cornejo etc., 23 Plant Mol.Biol.567:81,1993).To influence in the proteic transcription rate, the various factors well known in the prior art comprise the adjusting sequence at the processing botanical system, may be all influential as forward or negative role sequence, enhanser and silencer and chromatin Structure.The invention provides at least a interest albumen of expressing that can in engineered plant, utilize in these factors.Adjusting sequence of the present invention is natural genetic elements, promptly from isolating genetic elements the adorned selected plant.

External source: " external source ", concerning nucleic acid, mean nucleic acid derive from non-plant biological or derive from and plant will plant transformed not of the same race or be not derive from not with will the interfertile plant that does not belong to the target plant species of plant transformed.

According to the present invention, foreign DNA or RNA representative naturally occurring nucleic acid in the genetic composition of fungi, bacterium, virus, Mammals, fish or bird, but be not natural be present in will plant transformed in.Therefore, exogenous nucleic acid is to encode for example not by the nucleic acid of the polypeptide of the natural production of plant transformed.Exogenous nucleic acid needn't the proteins encoded product.According to the present invention, the transgenic plant of expectation are not contain the plant that is incorporated into any exogenous nucleic acid in its genome.

In yet another aspect, natural genetic elements can be mixed and is incorporated in the genome of the selected plant species according to the present invention.Be subordinated to selected plant species plant or from the affine plant of selected plant species the separating natural genetic elements.For example, be incorporated into n DNA in the potato of cultivation can derive from any genotypic potato or with potato (for example, S.demissum) the affine any genotypic wild-type potato seed of property.

Gene: " gene " is meant the coding region, do not comprise 5 '-or 3 '-hold the nucleotide sequence in this district.Functional gene is the coding region that is operably connected on promotor or the terminator.

Heredity is reset: being meant can be in vivo and the reconfiguring of external abiogenous genetic elements, and it has introduced the new organization of genetic stocks.For example, but the splicing each other of the polynucleotide of spontaneous generation on the locus of coloured differently body in vivo between development of plants and property recombination phase.Correspondingly, the reorganization of the external genetic elements that forms by non-natural genetic modification technology with also can be close by the recombination event of property reorganization generation in the body.

In-frame: the aminoacid sequence of nucleotide triplet (codon) being translated into the new life of the recombinant protein in the vegetable cell of expectation.Particularly, the present invention includes with reading frame and link first nucleic acid on second nucleic acid, wherein first nucleotide sequence is a gene, and second nucleic acid is promotor or similar regulatory element.

Integrate: be meant selected plant species, or the plant mutually of the same race, or be inserted in the genome of selected plant species cell with the nucleotide sequence of the affine plant of selected plant species with selected plant." integration " only is meant natural genetic elements is incorporated in the genome of vegetable cell.In order to integrate natural genetic elements, as passing through homologous recombination, the present invention can " use " dDNA as the step in this quadrat method.Therefore, the present invention distinguish " use " specific dna molecular and with specific dna molecular " integration " in the genome of vegetable cell.

Introduce: as used herein, be meant by the method that comprises infection, transfection, conversion or transduction a kind of nucleotide sequence is inserted in the cell.

Isolating: " isolating " is meant physically any nucleic acid or the compound of separating, the natural surroundings normal from it.Isolating material can be maintained in the suitable solution that contains for example solvent, damping fluid, ion or other composition, can be form purifying or non-purifying.

Leader sequence: (or 5 ' end) is transcribed but the sequence that is not translated before gene.

The LifeSupport carrier: the LifeSupport carrier is a kind of effable selected marker of containing between T-DNA or T-DNA sample edge, as the construction of neomycin phosphotransferase mark.Can modify the LifeSupport carrier to limit such mark and other polynucleotide between edge or edge sample sequence are integrated in the Plant Genome.For example, the LifeSupport carrier can comprise that virD2, the codA ∷ upp of sudden change merge, or any combination of this genetic elements.Therefore, the virD2 albumen of modification will still support T-DNA to transfer to plant nucleolus, but will limit efficient (Shurvinton etc., Proc Natl Acad Sci USA, 89:11837-11841,1992 of the genome conformity of T-DNAs subsequently; Mysore etc., Mol Plant Microbe Interact, 11:668-683,1998).Perhaps, before regeneration, can use codA ∷ upp gene fusion as negative selected marker.In a preferred construction, the LifeSupport carrier comprises the npt mark that is operably connected on the yeast ADH termination element.

Monocotyledons: its embryo has the flowering plant of a cotyledon.Monocotyledonous example comprises and singly is not limited to turfgrass, corn, paddy rice, oat, wheat, barley, jowar, orchid, Herba Iridis japonicae, lily, onion and palm.

Natural: " natural " genetic elements is meant that natural be present in, derive from or belong to will be by the genomic nucleic acid of plant transformed.Therefore, from will plant transformed or the genome of plant species isolating, perhaps from will transform that isolating any nucleic acid, gene, polynucleotide, DNA, RNA, mRNA or cDNA molecule are " natural " affine or interfertile plant of plant species or the plant species, promptly be the plant species inherent.That is to say that natural genetic elements is represented understandable all genetic stockss that are used for improving by the traditional plant breeding plant of plant breeder.According to the present invention, any variation of natural nucleotide also is considered to " natural ".Aspect this, " natural " nucleic acid also can separate from plant or the affine plant species of its property, and is modified or the variant that obtains of suddenling change lists with isolating not adorned natural acid from plant at nucleotides sequence and is greater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, or 60% similarity.The natural acid mutation lists also to have at nucleotides sequence and is less than approximately 60%, is less than 55%, or is less than 50% similarity.

Also the encode variant of the naturally occurring protein product from this nucleic acid, transcribing and translate of isolating from plant " natural " nucleic acid.Therefore, the natural nucleic acid native protein with unmodified expressed in the plant in isolating nucleic acid source of may encoding is greater than or equal to 99% on aminoacid sequence, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, or the albumen of 60% similarity.

Naturally occurring nucleic acid: this phrase meaning is that nucleic acid is present in the genome of selected plant species, and may be dna molecular or RNA molecule.The restriction enzyme site that exists normally can be transformed into ectogenic dna molecular in the genome of plant species, as carrier or oligonucleotide, even this restriction enzyme site is not isolating from this genome physically.Therefore, the present invention allows the synthetic nucleotide sequence that produces, as the Restriction Enzyme recognition sequence, as long as in the natural genome that is present in selected plant species of this sequence, perhaps be present in the affine plant of the selected plant species that will transform in.

Be operably connected:, make their functionatings correctly of in vegetable cell, joining together by this way in conjunction with two or more molecules.For example, when promotor control texture gene transcription, promotor is operably connected on the structure gene.

P-DNA: according to the present invention, P-DNA (" DNA of plants ") separates from Plant Genome and at each end, or only at an end, comprises T-DNA edge sample sequence.Edge sample sequence preference and edaphic bacillus bacterial classification have at least 50%, at least 60% as the T-DNA edge sequence of agrobacterium tumefaciens or root of hair plant Zymomonas mobilis, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95%, but less than 100% sequence identity.Therefore, available P-DNAs replaces T-DNAs that the nucleotide sequence of edaphic bacillus is transferred in another polynucleotide sequence.Can modify P-DNA so that clone, and should a preferably not natural proteins encoded or a proteic part.The characteristics of P-DNA are that it contains the edge sequence that at least one is known as " P-DNA edge sequence " or " P-DNA edge sample sequence " at each end, and above-mentioned two terms can exchange.Definition referring to top " edge sequence " and " edge sample ".P-DNA also can be counted as " T-DNA sample " sequence, the definition of face as follows.

Plant: comprise angiosperm and gymnosperm such as potato, tomato, tobacco, clover, lettuce, Radix Dauci Sativae, strawberry, beet, cassava, sweet potato, soybean, corn, turfgrass, wheat, paddy rice, barley, jowar, oat, Oak Tree, eucalyptus, English walnut and palm.Therefore, plant can be a unifacial leaf or dicots.Speech used herein " plant " also comprises vegetable cell, seed, plant filial generation, no matter be sexual or the propagulum of asexual generation and any one offspring in these, as transplanting a cutting or seed.Vegetable cell comprises suspension culture, corpus callosum, embryo, meristem zone, callosity tissue, leaf, root, branch, gametophyte, sporophyte, pollen, seed and sporule.Plant may be in the various stages in ripening stage, and can grow in the liquid or solid substratum, perhaps grows at soil or in flowerpot, greenhouse or field suitable medium.The expression of the leader sequence of introducing in plant, tailer sequence or gene order may be of short duration or persistent." selected plant species " can be, but be not limited to any one species in these " plants ".

Precise breeding: be meant by with nucleic acid, as from selected plant species or from another plant identical with selected plant species or from the affine kind of selected plant species isolating natural gene and regulatory element introduce in the single vegetable cell with being stabilized, subsequently with the vegetable cell regeneration whole plants of these genetic modifications to improve the method for plant.Since not with the unknown or exogenous nucleic acid for good and all be incorporated in the Plant Genome the therefore identical genetic stocks that also obtains of technology utilization of the present invention by the conventional plant breeding.

Plant species: belong to the demonstration plant group of the various definite designations of some sexual compatibility at least.

Plant Transformation and cell cultures: broadly be meant the genetically modified plant cell and with its transfer in the suitable plant culture with keep, further growth and/or the method for further growing.

Reorganization: as used herein, this term broadly is described as the various technology of relying clone gene, can carry out dna sequencing and can produce protein product.As used herein, this term also is described as with the transgenosis albumen that the back is produced in the cell of plant host system.

Selected marker: " selected marker " encode typically can give certain to the resistance of microbiotic, weedicide or toxic compounds albumen and be used to identify the gene of transformation event.The example of selected marker comprises that coding have streptomycin phosphotransferase (spt) gene of resistance to strepto-, Man-6-P is changed into phosphomannose isomerase (pmi) gene of fructose-6-phosphate, neomycin phosphotransferase (nptII) gene of coding kantlex and Geneticin resistance, coding has hygromix phosphotransferase (hpt or the aphiv) gene of resistance to Totomycin, coding has acetolactate synthase (als) gene of resistance to sulfonylurea herbicide, coding (for example suppresses glutamine synthetase such as phosphine silk mycin or basta, the bar gene) effect has the gene of resistance, other similar gene perhaps well known in the prior art to weedicide.

Have justice to suppress: the expression of one or more additional copy of all or part of gene by this gene in the transgenic plant reduces the expression of native gene.

The T-DNA sample: " T-DNA sample " sequence is from selected plant species, or from the affine plant of selected plant species isolating nucleic acid, the T-DNA of itself and edaphic bacillus kind has at least 75%, 80%, 85%, 90%, or 95%, but be not 100% sequence identity.T-DNA sample sequence can comprise one or more edge or edge sample sequence, and wherein each sequence can both be incorporated into nucleotide sequence in another polynucleotide." P-DNA " used herein is an example of T-DNA sample sequence.

Tailer sequence: (or 3 ' end) transcribes but the sequence do not translated after gene.

The DNA that transcribes: comprise gene and with the leader sequence of the untranslated of this gene-correlation and the DNA of tailer sequence, it is transcribed into single mRNA the effect of the promotor by the front.

Transcribe and translation termination: expression vector of the present invention typically has transcription termination region in the opposite end of transcription initiation regulatory region.Can select transcription termination region to be used for stable mRNA to improve expression and/or to be used for adding poly A tail to gene transcription product (Alber; Kawa saki, Mol.﹠amp; Appl.Genetics 4:19-34,1982).Comprise the E9 sequence (Mogen etc., Mol.CellBiol., 12:5406-14,1992) of pea RBCS gene and the termination signal of various ubiquitin genes as illustrational transcription termination region.

The conversion of vegetable cell: DNA stably is incorporated into the method in the genome of vegetable cell." stably " be meant persistent or non-of short duration reservation and/or expression in the cellular genome and that pass through the polynucleotide of cellular genome.Therefore, stably the polynucleotide of Zheng Heing be a kind of in the cell transformed genome polynucleotide as fixture, and the filial generation in succession of conversion plant that can be by cell or gained is replicated and breeds.Conversion can be under natural condition or is used under the artificial condition of various known methods in the prior art and take place.Conversion may depend on nucleotide sequence is inserted into any currently known methods in protokaryon or the eukaryotic host cell, comprises agrobacterium-mediated conversion scheme, virus infection, whisker phage, electroporation, heat-shocked, lipofection, polyoxyethylene glycol processing, microinjection and particle bombardment.

Transgenosis: will be inserted into the gene that comprises protein-coding region in the host genome.In the context of the present invention, separation comprises genetically modified element from host genome.

Transgenic plant: the plant that contains at least one genetically modified genetic modification.

Use: the present invention imagines the nucleic acid that uses the species that are different from the selected plant species that will transform so that natural genetic elements is incorporated in the genome of selected plant, as long as such exogenous nucleic acid is not incorporated in the identical host plant gene group with not being stabilized.For example, natural genetic elements cloned, located or be manipulated to wherein plasmid, carrier or clone's construction can from the natural different species in genetic elements source.

Variant: " variant " used herein should be understood that to mean Nucleotide or the aminoacid sequence that departs from Nucleotide specific gene or proteic standard or given or aminoacid sequence.Term " isoform ", " isotype " and " analogue " also refer to " variation " form of Nucleotide or aminoacid sequence.By one or more amino acid whose interpolations, remove or replace the aminoacid sequence that changes or the variation of nucleotide sequence, can be considered to " variant " sequence.Variant can have " guarding " and change, and wherein the amino acid of Qu Daiing has similar structure or chemical property, for example, replaces leucine with Isoleucine.Variant can have " nonconservative " and change, and for example replaces glycine with tryptophane.Similar less variation also can comprise amino acid whose disappearance or insertion, and perhaps the both has.Use computer program commonly known in the art, as carrier NTI package (InforMax, MD) software can find the decision which amino-acid residue can be substituted, insert or disappearance aspect guidance.

These therefore it should be understood that to the invention is not restricted to specific method described herein, scheme, carrier and reagent etc., because can change.Should be appreciated that also term used herein only is used to describe the purpose of particular, does not limit the scope of the present invention.Must notice here and " one " of the singulative that in appending claims, uses that " one " and " described " comprise the plural number relation, unless context clearly requires other implication.Therefore, for example, mention that " gene " is meant and relate to one or more genes, comprise its equivalent well known by persons skilled in the art etc.In fact, those skilled in the art can use method described herein to express any natural gene (present or known subsequently) in the plant host system.

The P-DNA carrier

The preferred method that recombinant DNA is incorporated in the vegetable cell is agrobacterium-mediated method for transformation.According to the present invention, the two carriers of exploitation are to produce the potato plant of the genetic modification that only contains natural potato nucleic acid.This carrier is different from conventional agrobacterium-mediated conversion carrier aspect three: (1) carrier of the present invention does not contain the T-DNA sequence in the edaphic bacillus source of describing at useful T-DNA edge, and contain DNA (P-DNA) fragment that both sides are the natural phant of edge sample sequence, although edge sample sequence is different from the T-DNA edge on 26S Proteasome Structure and Function, but they support P-DNA is transferred to the vegetable cell from edaphic bacillus, (2) main chain of carrier of the present invention can contain underlined, if be integrated in the genome of vegetable cell, this mark can stop these cell developments to become sophisticated plant, and (3) carrier of the present invention does not contain the selected marker of external source between the P-DNA end.

The present invention shows that astoundingly both sides are that the P-DNA fragment of edge sample sequence supports that DNA transfers to the vegetable cell from edaphic bacillus.By using, can from the genome of any plant, separate P-DNA based on the designed primer of homology between potato P-DNA end and the conventional T-DNA edge.Then check these fragments,, cut to pieces and be used to transform the plant that only contains n DNA if effectively.Also can service routine, search for the Plant Genome database as " blastn " and contain the dna fragmentation (Altschul etc., J Mol Biol 215:403-10,1990) that has the zone of homology with the T-DNA edge with searching.The P-DNAs that then can modify the process evaluation is to improve their effectiveness.For example, the interior segments that can delete isolating P-DNAs also can be added restriction enzyme site so that the clone.Sequence introducing point mutation also may be that effectively it makes P-DNA more effective in transfer DNA endways.

Can between P-DNA edge sample sequence, insert any expression casette.For the conversion of potato, such expression cassette can by the gene that is operably connected to potato and/or with the leading or tailer sequence of this gene-correlation on the potato promotor and potato terminator subsequently constitute.Expression cassette can contain other potato genetic elements, as the in-frame signal peptide sequence that is fused to gene 5 '-end, and can place, for example the potato intron of expressing with raising between promotor and the interest genes.For with the P-DNA transformed wheat of modifying, all genetic elements of in wheat P-DNA, inserting, comprise P-DNA itself all from wheat or with the affine plant species of wheat.

Another method of separating P-DNAs is by generation edaphic bacillus bacterial strain library, and its DNA of plants fragment rather than both sides of containing at random are selected marker's T-DNA.The explant that infects with this library can be placed on the proliferated culture medium that contains suitable selectivity reagent, support the carrier of marker gene from edaphic bacillus transferred to P-DNAs in the vegetable cell to identify.

The P-DNA of natural modifications not only during conversion process, and other plasmid sequence is also transferred to the vegetable cell jointly from edaphic bacillus.For purpose of the present invention, this is one and does not expect the process wanted, because such plasmid " main chain " sequence is represented the foreign DNA of non-plant, as DNA of bacteria.The present invention stops the plant transformed cell development that contains the main chain sequence to form sophisticated plant.Therefore, the invention enables the transformation event that difference contains main chain and do not contain main chain during the regeneration branch to become possibility.

Select or screen the method for the incident that anti-main chain integrates to depend on the main chain of carrier, outside P-DNA, whether have mark, as isopentene phosphotransferase (IPT) expression of gene box.In case main chain has been integrated, the accumulation of IPT inductive phytokinin will change the proterties of the branch that transforms, and stop these shoot growths to go out root.Can use any other gene of proterties, quality or color, height or some other morphological specificity of the leaf that can change plant transformed, root, stem to replace the IPT gene to be used to the incident of screening and/or selecting anti-main chain to integrate.Such gene is called as " main chain integration mark " here.Therefore, known demonstration be attributable to change that the main chain integration marker gene expresses the conversion plant of morphological specificity except the P-DNA that contains expectation, in its genome, also contain foreign DNA.Correspondingly, the plant with phenotype relevant with main chain integration mark is not expected to want.

The invention is not restricted to only use the IPT gene to integrate mark as main chain; Can use other gene by this way.For example, it can be anti-zeatin (transzeatine) synthase (TZS) gene (Krall etc. of edaphic bacillus that main chain is integrated mark, FEBS Lett 527:315-8,2002) or recessive Arabidopsis (Arabidopsis) gene hocl (Catterou etc., Plant J 30:273-87,2002).In the present invention, this method is easier to use than some methods of inserting virulent gene in carrier main chain sequence.For example, referring to EP 1009,842.

By with the main chain integration marker gene, place upstream or the downstream of P-DNA as functional cell mitogen gene, be easy to distinguish transformation event.The transformed plant of morphological character that abandoned display change is because they contain the non-natural dna sequence dna that is incorporated in the genome.

Another kind of only identify that the strategy with the plant of the stable conversion of n DNA is to use polymerase chain reaction.By using specificity to design the primer that detects the main chain sequence, can identify and abandon the plant that except P-DNA, also contains external source main chain sequence.Can use other primer sets whether intactly to shift P-DNA subsequently with further confirmation.Therefore, by the morphological character of use expression of gene, or, can identify and select only to use the plant of natural DNA sequence stable conversion by screening the foreign DNA of stable integration in transforming plant with the change plant.

The genetic elements of specific host plant can be inserted in the P-DNA sequence of two carriers that can in intestinal bacteria and edaphic bacillus, duplicate.Can the carrier that obtain be incorporated in (disarmed) the edaphic bacillus bacterial strain such as LBA4404 of being laid down one's arms by chemoreception attitude cell being carried out electroporation, three parental plant mating or heat-shocked handle.Then can new bacterial strain be used to transform one vegetable cell by the infection of whole plant or explant.

The genetic elements of specific host plant can be inserted in the P-DNA sequence of two carriers that can in intestinal bacteria (E.coli) and edaphic bacillus, duplicate.Can the carrier that obtain be incorporated among edaphic bacillus bacterial strain such as the LBA4404 by chemoreception attitude cell being carried out electroporation, three parental plant mating or heat-shocked handle.Then can new bacterial strain be used to transform one vegetable cell by the infection of whole plant or explant.LBA4404 contains the Ti-plasmid pAL4404 that is laid down one's arms, and it has toxic action gene and streptomycin resistance gene.

The LifeSupport carrier

Although the bacterium marker gene stably is incorporated into the evaluation of being convenient to transformation event in the genome of vegetable cell, such modification of Plant Genome is not expected to want, because marker gene is represented foreign DNA.Can avoid using the marker gene of external source by developing new method for transformation based on edaphic bacillus.

An embodiment preferred is to rely on a kind of novel method of using two edaphic bacillus bacterial strains: a bacterial strain contains the two carriers with the selected marker who is used for transient expression in plant nucleolus, and another bacterial strain contains the P-DNA (seeing embodiment 7) of the interest sequence with the reality that is used for stable integration in Plant Genome.

In case carried out cotransfection with the edaphic bacillus bacterial strain, so some vegetable cells P-DNA that will receive T-DNA and have the interest sequence with marker gene.Do not place suitable microbiotic to come the stable integration of selectable marker gene for a long time by the explant that will infect subsequently, but only explant is exposed in the microbiotic momently.Like this, the vegetable cell of the gene of the mark of all transient expressions will be survived.Because because the activity of endogenous nucleic acid alcohol, T-DNAs will degrade rather than stably be incorporated in their hosts' the genome in most examples, all develop into the branch that lacks marker gene so be presented at most of vegetable cells of surviving in the of short duration selection here.In addition, the present invention shows that the considerable part in these unmarked branches contains the P-DNAs of stable integration.

The instrument that the unmarked transformation efficiency of many raisings is arranged.At first, the present invention shows and can increase this frequency by the soil bacillus strain infection explant that has T-DNA/ mark and P-DNA/ interest sequence respectively with two.At first use P-DNA strain infection explant, use the T-DNA strain infection after about 4 to 6 hours.

The second, can modify the T-DNA bacterial strain to express the virD2 gene of Q-spoiling.The virD2 albumen of modifying will still be supported T-DNA is transferred in the plant nucleolus, but efficient (Shurvinton etc., Proc Natl AcadSci USA, 89:11837-11841,1992 of the genome conformity of restriction T-DNAs subsequently; Mysore etc., Mol Plant MicrobeInteract, 11:668-683,1998).The method of the virD2 gene that most preferred expression is modified is by insert the virD2 gene of the Ω sudden change that is subjected to the vitD promoters driven in the main chain of T-DNA carrier.

The 3rd, further reduce stable T-DNA near left hand edge that can be by telomeric sequence being inserted T-DNA and the right hand edge sequence and integrate (Chiuazzi ﹠amp; Signer, Plant Mol.Biol., 26:923-934,1994).

The 4th, the size that can increase the T-DNA district that has marker gene is with the frequency in raising T-DNAs and the common shift-in plant nucleolus of P-DNAs, and the frequency of the genome conformity of minimizing T-DNA.

The 5th, also can improve the frequency in T-DNAs and the common shift-in plant nucleolus of P-DNAs by the single edaphic bacillus bacterial strain that two two carriers of the affinity that has T-DNA and P-DNA are respectively carried in use.The example of two carriers of two affinities is the carrier in pSIM 1301 sources and the carrier in pBI121 source.

Because the marker gene of transient expression usually with unconformability in Plant Genome, so needn't this gene and its regulate sequence both and represent n DNA.In fact, using external source to regulate sequence may be favourable with high-caliber of short duration genetic expression in the vegetable cell that promotes to infect.Wonderful discovery of the present invention is to contain gus gene, follows expression cassette high level ground transient expression in potato cell by Alcohol Dehydrogenase from Yeast 1 (ADH1) terminator.Yet the similar construction with yeast CYC1 terminator can not work fully.Also can improve of short duration expression level on non-natural promotor by marker gene is operably connected to.The example of such promotor is, for example the promotor of synthetic promotor such as glucocorticoid inducible (Mori etc., Plant J., 27:79-86,2001; Bohner etc., Mol.Gen.Genet., 264:860-70,2001), the 35S promoter of non-natural promotor such as cauliflower mosaic virus and figwort mosaic virus, and the promotor of fungi.

As Gong the alternate method of the agrobacterium-mediated method for transformation of above-described use two strains, also can transform plant with the single bacterial strain that comprises P-DNA with natural marker gene and interested actual sequence.The present invention shows can use the natural mark of salt tolerance gene as conversion.Such salt tolerance gene comprises Arabidopsis gene SOS1 (Shi etc., Nat Biotechnol., 2002), AtNHX1 (Apse etc., Science, 285:1256-8,1999), Avpl (Gaxiola etc., Proc Natl Acad Sci USA, 98:11444-9,2001) and CBF3 (Kasuga etc., Natl Biotechnol.17:287-91,1999) farm crop homologue.

The rearrangement of the genetic elements of finishing by precise breeding methodology of the present invention also can spontaneously take place by the method for genetic recombination.For example, all plants all contain can be from a chromosome position swivel base to another chromosome position element.By being inserted in promotor or the gene, such transposable element can improve, changes and/or reduce expression of gene.For example, in the promotor of transcriptional regulatory gene P-wr, insert the AMu4 that corn increases the argument part and cause streaky haw skin.In the promotor of the specific MADS box of leaf gene ZMM19, insert components identical and cause the expression of this gene in the corn inflorescence, cause the lobate prolongation of lepicena and other change in the male and female inflorescence, cause the very gratifying phenotype of beans English cereal.Because its peculiar male flower flower fringe and grain ear, beans English cereal has religious significance for some indigenous America clan.Many genes are also modified by other transposon inductive and are reset as inversion, disappearance, interpolation and dystopy.(Bennetzen，Plant Mol Biol42：251-69，2000)。In addition, DNA of plants is reset and is often taken place by intragenic regrouping process.For example, by related gene in the resistance that is binned in anti-special pathogenic agent, plant can produce has new specific resistant gene, and therefore with their pathogenic agent coevolution (Ellis etc., Trends Plant Sci 5:373-9,2000).Another example of recombinating in the gene relates to plant and how to breed: make plant carry out the transition to selfing (Kusaba etc., Plant Cell13:627-43,2001) from cross pollination by being binned in the gene that relates in the self incompatibility.Other promotes the method for genome evolution to comprise, for example, and rhexis and interchromosomal reorganization.

Improve the nutritive value of plant and food crop

In order to modify the passive characteristic of farm crop by precise breeding, as acrylamide accumulation, glycoalkaloid accumulation, the accumulation of undesired advanced glycation product, the accumulation of CIPC, low-level Resistant starch, scar susceptibility, cold inductive during the processing sweeten, susceptibility, low yield and inferior quality to disease, at least a specific expression cassette is incorporated in host's the genome.Use three kinds of diverse ways to remove passive characteristic: the gene that (1) overexpression stops passive characteristic to take place, (2) for albumen the gene product titration of wild-type is come out with non-functional, the mutant form of the gene that overexpression is relevant with passive characteristic, and (3) make the specific gene silence relevant with passive characteristic by expressing and segmental at least one copy of the leading or tailer sequence on justice and/or antisense orientation of this gene-correlation.

In potato relevant with passive characteristic and external can be made example of its coding NOT-function proteic native gene by modification be polyphenoloxidase (PPO) gene.In case be subjected to impact injury, the PPO gene product that discharges from plasmid enters tenuigenin (Koussevitzky etc., J.Biol.Chem., 273:27064-9,1998), wherein this gene product is led joint the oxidation of phenol and is produced various phenoxy group free radicals and quinoid derivative, and these materials are that the polymkeric substance that deleterious and/or final formation is wanted stays dark variable color in farm crop, or " blackspot ".

The sudden change PPO gene that overexpression contains non-functional structural domain in conjunction with copper can reduce mainly the activity of all PPO genes of expressing in stem tuber and relevant organ such as bud.Sudden change makes polyphenol oxidase zymoprotein inactivation, because it can not be in conjunction with copper.Where the technician knows is carried out point mutation, if like this, that will damage the function of gene product.The applicant is by comparing the structural domain of having identified among the potato PPO in conjunction with copper (Klabunde etc., Nat Struct.Biol., 5:1084-90,1998) with PPO protein sequence of potato and the protein sequence of sweet potato PPO.The target that conservative zone, particularly those zones of containing conservative Histidine on copper binding site is render transgenic product inactivations.Almost completely lack the ability negatively to influence plant opposing pathogenic agent because PPO is active in such organ, so the present invention has also described only to reduce improving one's methods of main specificity PPO gene of expressing except epidermis in all parts of ripe stem tuber.The PPO that the silence of this species specific PPO gene can not be reduced in the stem tuber epidermis owing to the tailer sequence by use and this gene-correlation expresses, and therefore this part with stem tuber almost all directly is exposed in the pathogenic agent of attempting to infect.

By the quality of PPO gene induced Enzymatic browning not only reduction potato tuber, it also negatively influences crop food such as wheat, avocado, banana, lettuce, apple and pears.

Relevant with passive characteristic and can be comprised R1 gene and L type phosphorylase gene by other reticent gene by using with the leading or tailer sequence of these gene-correlations.Become reducing sugar at starch degradation, as relating to this two genes in glucose and the fructose, in a single day reducing sugar is heated with regard to participating in Maillard reaction and produces toxic products such as acrylamide.The present invention shows, reduces cold inductive by the activity that reduces R1 or Starch phosphorylase and sweetens and cause reducing the non-Enzymatic browning during the fried processing of potato and the accumulation of acrylamide.

The present invention also shows the purposes of some natural gene of overexpression in the crop of genetic modification.Reduce sucrose to conversion of reduced sugar by the new isolating vacuolus converzyme inhibitor gene of overexpression in potato, reduced the level of maillard reaction product such as acrylamide significantly.

The present invention also foretells and show that the potato tuber that converting enzyme inhibitor expression level raising or R1 or Starch phosphorylase expression level reduce will need thoroughly not handle with chemical sprout inhibitor such as CIPC before storage, because postponing (1), the reducing sugar level that they reduce germinates, (2) allow under lower temperature, to preserve, thereby further postpone germination.Extremely CIPC residual level that reduces or shortage have further improved the nutritive value of the processed food of the P-DNAs plant origin that contains above-mentioned certain modification.

Therefore, derive from the French fried food product of the P-DNA stem tuber that contains modification or the CIPC residual level that chips will contain violent reduction, further improve their nutritive value.

Reduce the influence that PPO in the potato tuber and R1 or phosphorylase gene express simultaneously and work in coordination with, because the non-Enzymatic browning not only by Maillard reaction, and the brown stain of PPO enzyme mediation also needs reducing sugar.Therefore, the reduction of reducing sugar level also will limit the active of PPO and to the susceptibility of blackspot scar in the transgenic Rhizoma Solani tuber osi stem tuber.Therefore, PPO, R1 and phosphorylase gene and/or with the representative of the leading or tailer sequence of these gene-correlations can be separated, modify and introduce back again in the plant to reduce the interest dna fragmentation of these expression of gene.

Except cold the inducing of developing scar resistance and reduction sweetens, can introduce many other characteristics by the precise breeding that does not use foreign DNA.For example, can from the wild-type potato seed, separate in the resistant gene and the genome of insertion of disease the kind of disease-susceptible humans.

Plant of modifying and the environmental benefit of crop

As described above, the level of R1 or Starch phosphorylase reduces the reduction that causes starch phosphorylation.The reduction of this starch phosphorylation causes the phosphorus acid content of potato tuber to reduce by 90% (Vikso-Nielsen, Biomacromolecules, 2:836-43,2001).This will cause the reduction of the phosphoric acid level in the potato processing factory waste water, and it is about 25-40mg/L now.Therefore, use the stem tuber of low phosphoric acid that minimizing is discharged into the phosphoric acid in the environment and helps to protect the important ecosystem.In addition, for the growth and the output of the best, low phosphoric acid potato may need with less phosphoric acid fertilising, its by the consumption support of postponement phosphoric acid resource can be more lasting agricultural.

Improve the agriculture characteristic of plant and food crop

Except two important processing characteristics, promptly reduce outside scar susceptibility and cold the sweetening, the present invention also provides important gradually input characteristic, i.e. salt tolerance.The P-DNA construction of some modifications of Miao Shuing in the present invention contains the salt tolerance gene as the natural mark that transforms.Importantly be that the effectiveness of this gene is not limited to the screening step in the method for transformation.The overexpression of salt tolerance gene can reduce high salinity soil level institute inductive and coerces symptom in the potato plant, and will make the new variety that on the farm land of saltness horizontal exceeding maximum value 2 mmhos/centimetre level of conductivity that percentage is increasing, grow the P-DNA that contains modification become possibility, this level of conductivity is the optimum condition of growth conventional variety.

Use from selected plant species or from the affine kind of selected plant species isolating regulatory element

In case from interested plant species, isolate leader sequence, gene or tailer sequence and can randomly modify, it can be operably connected to the promotor of plant so or similarly be used for suitably expressing on the regulatory element plant.Regulatory element such as these are used to express non-translated sequence with interested gene-correlation in specific tissue or with certain level or in the specific time.

Depend on related strategy in modifying characteristic, may the essential silence that limits the specific region of plant.Normally order about the native gene expression promoter and may be not suitable for tissue-specific expression.As describing in the part in the above, bacterium or viral regulatory element can cause that can't estimate and undesired incident as the stable integration of 35S " super " promotor of cauliflower mosaic virus.Therefore, one aspect of the present invention uses isolating promotor from selected host plant kind.

In embodiment preferred of the present invention, to be operably connected to the leading or tailer sequence of R1, Starch phosphorylase and PPO gene-correlation on the promotor with particle bonded starch synthase gene, in order that in potato (S.tuberosum), use (Rohde etc., JGen﹠amp; Breed, 44,311-315,1990).This promotor often is used for driving genetic expression and at potato tuber activity (van der Steege etc., PlantMol Biol, 20:19-30,1992 is arranged especially by other people; Beaujean etc., Biotechnol.Bioeng, 70:9-16,2000; Oxenboll etc., Pro Natl Acad Sci USA, 9:7639-44,2000).In preferred embodiments, the expression of the leading or tailer sequence of R1, Starch phosphorylase and the PPO gene that also this promotor can be used to modify.

Perhaps, other potato promotor can be operably connected on the interest sequence of potato.Such promotor is included in the potato tuber protein gene promoter (Bevan etc. that promote expression in the potato tuber, Nucleic Acid Res, 14:4625-38,1986), or its fragment, potato UDP-glucose pyrophosphorylase gene promoter (U.S. Patent No. 5,932,783) and the promotor (Garbarino etc. of ubiquitin gene, PlantPhysiol, 109:1371-8,1995).

Also can regulate transcribing of leading and/or tailer sequence by the regulatory region that uses inducible promoter and in construction, operationally link on the polynucleotide of interest.The example of inducible promoter comprises those thermally sensitive promotors, as heat or cold shock promotor.For example potato ci21A-and C17-promotor are cold inductive (Kirch etc., Plant Mol Biol, 33:897-909,1997; Schneider etc., Plant Physiol, 113:335-45,1997).

Can use some material is resembled other inducible promoter that microbiotic, other chemical substance or pH reply.For example, known dormin and gibberic acid influence (and in so doing) such as internal pHs of vegetable cell, regulate Rab 16A gene and αDian Fenmei 1/6-4 promotor (Heimovaara-Dijkstra etc., Plant Mol Biol, 4815-20,1995).Known dormin, wound and methyl jasmonate are also induced potato pin2 promotor (Lorberth etc., Plant J, 2:477-86,1992).

In another example, some nucleotides sequences are listed under the sequential control, and only certain time durations during certain etap of plant or in a day is activated and expresses downstream sequence.For example, ribulose-1,5-bisphosphate, the potato promotor of the little subunit of 5-bisphosphate carboxylase (rbcS) gene can instruct the expression (Fritz etc., Proc Natl AcadSci USA, 88:4458-62,1991) of the light regulation and control of cell-specific.The technician is proficient in inducible promoter and regulates these typical types of sequence very much.

The stability of using some polyadenylation signal to change the mRNA transcript in regulating expression may also be effective.Particularly, when being operably connected to 3 ' end of polynucleotide, some polyadenylation signals cause the mRNA transcript to be easy to degraded.

Therefore, can be by it operationally be connected and the expression of regulatory gene with one or more such promotor, adjusting sequence, 3 ' polyadenylation signal, 3 ' non-translational region, signal peptide etc.According to the present invention, as described herein those, and to be integrated into dna sequence dna in the Plant Genome and regulatory element the most at last all be to obtain from the DNA of the selected plant species that will modify by precise breeding method of the present invention.That is, be derived from, separate from and the clone from other species, be not integrated in the genome that transforms plant as the dna sequence dna of bacterium, virus, microorganism, Mammals, birds, Reptilia and the incompatible plant species of property and regulatory element.Only otherwise this foreign DNA is incorporated in the Plant Genome, can use in the present invention with the allogenic DNA of the genome of selected plant species and transform construction to produce.

The present invention not only provides by integrating from selected plant species, or from the affine plant of selected plant species the DNA that obtains transform the method for plant species, and the method that provides adjustable this DNA to express.Correspondingly, can pass through as described previously, tissue-specific or a certain other strategy is optimized the expression of certain sequence.

Use is isolating terminator sequence from selected plant species

Except the regulatory element that initiation is transcribed, natural expression cassette also needs to be positioned at the element that the termination of initial the regulatory region 3 '-end of transcribing is transcribed.Can obtain transcription termination region and transcription initiation region from homologous genes or from different genes.Can select to be particularly useful for stable mRNA to strengthen the transcription termination region of expressing.

This special element, promptly so-called " 3 '-non-translational region " is very important in transhipment, stable, location and terminator are transcribed.Aspect this, the known 3 '-non-translational region of those skilled in the art can form certain hairpin loop.Correspondingly, the present invention anticipation can be operably connected to 3 '-non-translational region 3 ' end of the polynucleotide of being cloned, so that the mRNA transcript that obtains can be exposed in the factor that the sequence of being given by 3 ' non-translational region and structure are worked.

Can be from isolate promotor and genetically modified plant species 3 ' sequence of subclone ubiquitin gene, and be inserted into of the suitable termination of genetically modified downstream to guarantee to transcribe.Which promotor that don't work is ordered about their expression, two typical transgenosiss can be fused on the terminator sequence of potato ubiquitin gene (Ubi3).

Embodiment

Embodiment 1

The clone of P-DNAs

This embodiment shows that T-DNA edge sequence is special to edaphic bacillus.This embodiment shows that also plant contains T-DNA edge sample sequence, and the sequence of the isolating dna fragmentation of describing by such edge sample sequence from potato and wheat is provided.

The T-DNAs that conventional conversion system uses the edaphic bacillus source is as foreign DNA is transferred to carrier (Schilperoort etc., United States Patent (USP) 4940838,1990) the vegetable cell from edaphic bacillus.Although T-DNAs generally includes a hundreds of base pair, to describe with left hand edge (LB) and right hand edge (RB) tumor-necrosis factor glycoproteins, they may also only be made of such edge sequence.T-DNA edge sequence plays an important role in the DNA transfer process, because they play a part the specific recognition site as the catalytic cleavage reaction of virD2-.The single stranded DNA that will form the release of complex body with the virD2 and the virE2 of edaphic bacillus is transferred in the plant nucleolus that often successfully is incorporated in the Plant Genome.Be used for all T-DNA edge sequences that foreign DNA shifts all from the nopaline and the octopine bacterial strain (table 2) of agrobacterium tumefaciens and root of hair plant Zymomonas mobilis.These edge sequences and some sequences that often are arranged in edaphic bacillus (Agrobacterium) DNA both sides are present in thousands of two carriers, these pairs carrier comprises, pPAM (AY027531) for example, pJawohl (AF408413), pYL156 (AF406991), pINDEX (AF294982), pC1300 (AF294978), pBI121 (AF485783), pLH9000 (AF458478), pAC161 (AJ315956), BinHygTOp (Z37515), pHELLSGATE (AJ311874), pBAR-35S (AJ251014), pGreen (AJ007829), pBIN19 (X77672), pCAMBIA (AF354046), pX6-GFP (AF330636), pER8 (AF309825), pBI101 (U12639), pSKI074 (AF218466), pAJ1 (AC138659), pAC161 (AJ315956), pSLJ8313 (Y18556), and pGV4939 (AY147202).Recently, two homologues (Palanichelvam etc., Mol Plant Microbe Interact 13:1081-91,2000) of T-DNA edge sequence in chrysopine type Ti-plasmids pTiChry5, have been identified.The left hand edge homologue is identical with the edge homologue of the T-DNA intermediary non-activity that is positioned at pTi15955.The right hand edge homologue deviates from the edge sequence of function T-DNA unusually.Therefore these homologues are transferred to DNA the vegetable cell from pTiChry5 in support and can not be had functionally active.

Only need to make the conversion plant become the exploitation of possible novel method, at first need to replace the T-DNA that comprises LB and RB with n DNA.Unfortunately, comprising that those middle-and-high-ranking blast searches of public database of being safeguarded by NCBI (National Center For Biotechnology Information), Joint Genome Institute (The Institute for Genomic Research) and SANGER fail to identify any edge sequence of plant.Therefore, the edge sequence similarity of essential consideration and T-DNA, but incomplete same DNA of plants sequence is called " edge sample " (edge sample) sequence here.Be presented at the example of the plant edge sample sequence that identifies in the public database in the table 2.The proposal of attempting with edge sample sequence replacement T-DNA edge sequence is high conservative (seeing Table 2) because of the edge sequence.Transfer system such as IncP, PC194 in other DNA of bacteria, and the major part of these sequences also is a high conservative in the cutout regions of φ X174, show that these sequences are to put together sample DNA to shift necessary (Waters etc., Proc Natl Acad Sci 88:1456-60,1991).Owing to do not have reliable data on about the requirement to the edge sequence, as if therefore whole edge sequence is very important in cutting process.The single research of attempting by checking the efficient of edge mutant in supporting the DNA transfer to be devoted to this problem is insecure, (the van Haaren etc. because as if negative control do not work rightly, Plant Mol Biol 13:523-531,1989).In addition, none is proved on molecules in the result of this research.Although these worries are arranged, two effectively edge mutant also have been displayed in Table 2.

In the edge sequence,, identified T-DNA edge motif (table 2) based on homology.Although this motif comprises 13,824 variants, many variants in these variants may not have function in transfer DNA, perhaps may be unsuitable, but to represent T-DNA edge sequence be what maybe may be the most wide in range possible definition of what sequence for it.Then in the DNA of public Ke De database, use " motif contrast and research tool " (Bailey and Gribskov with this edge motif, Bioinformatics 14:48-54,1998) and " senior BLASTN " (" Nucleotide mispairing point penalty "=-1; " expected value "=10 ⁵Altschul etc., Nuclei Acids Res 25:3389-3402,1997) the search homologue.These search fail to identify any identical coupling once more in the biology of non-agrobacterium.

In order to attempt and to improve the probability of isolating the potato dna fragmentation that contains the edge sample sequence that is equivalent to the edge motif, (so-called " core collection " provided by U.S. potato gene storehouse, isolates DNA in WI) from the different registration number of 100 heredity.This DNA is a blended, and is used as the template of the polymerase chain reaction that uses various oligonucleotide, and wherein oligonucleotide is designed to be annealed on edge or the edge sample sequence.Fragment to amplification is carried out sequential analysis, then uses inverse PCR further to confirm sequence with nested primers.An interested especially fragment contains the new sequence that has or not any main open reading-frame (ORF) in the potato dna fragmentation, and it is depicted as edge sample sequence (table 2).One of this segmental edge sample sequence contains at least 5 mispairing with T-DNA edge sequence; Another edge sample sequence contains at least 2 mispairing.Although two sequences all contain a mispairing with the edge motif, still to check their to support the ability that DNA shifts.For that purpose, at first should segmental size reduce to 0.4 thousand base pairs (SEQ ID NO.:1) by the inside disappearance.The fragment that obtains is called as " P-DNA " (DNA of plants) to be different from the T-DNA in edaphic bacillus source.Separating analogous fragment from the genome of potato mutation RussetRanger, but be not used in any further experiment.

Based on the difference between P-DNA and the T-DNA edge sequence, use P-DNA:5 '-GTTTACANHNBNATATATCCTGYCA-3 ' that the extension enzymatic amplification system (Life Technologies) with following degenerated primer separates wheat (Bor-F) (SEQ ID NO.56) and 5 ' TGRCAGGATATATNVNDNTGTAAAC 3 ' (Bor-R) (SEQ ID NO.57).The fragment of 825 base pairs that obtain is shown as SEQ ID NO.:2, and is used to replace the T-DNA of conventional two carriers.Can pass through between the P-DNA end, to insert the gus gene expression cassette, and infect the efficient that wheat is checked this construction with the edaphic bacillus that contains the gained carrier (Agrobacterium).

Embodiment 2

With P-DNA carrier transformation of tobacco

This embodiment shows, although there is the difference of structure (sequence difference) and function (transformation frequency) between P-DNA end and the T-DNA edge sequence, can be to use P-DNA so that DNA is transferred to the tobacco cell from edaphic bacillus with the similar mode of T-DNA.

By remove conventional two carrier pCAMBIA1301 (Cambia, the no T-DNA carrier that whole T-DNA district acquisition AU) can be kept in intestinal bacteria and agrobacterium tumefaciens.This by simultaneously with the 5.9kb SacII-SphI fragment of pSIM1301 with use oligonucleotide that two fragments that increase from pCAMBIA1301 are connected to finish, wherein oligonucleotide is to being respectively: 5 ' CCGCGGTGATCACAGGCAGCAAC 3 ' (SEQ ID NO.58) and 5 ' AAGCTTCCAGCCAGCCAACAGCTCCCCGAC 3 ' (SEQ ID NO.59), and 5 ' AAGCTTGGCTACTAGTGCGAGATCTCTAAGAGAAAAGAGCGTTTA 3 ' (SEQ ID NO.60) and 5 ' GCATGCTCGAGATAGGTGACCACATACAAATGGACGAACGG 3 ' (SEQ IDNO.61).

For the incident that the anti-main chain of screening is integrated becomes possibility, to comprise by edaphic bacillus prenyltransferase (IPT) gene of Ubi3 promoters driven and thereafter the expression cassette of Ubi3 terminator (SEQ ID NO.:3) be inserted in the main chain of above-described no T-DNA carrier as 2.6kbp SacII fragment, produce pSIM100-OD-IPT.Expection is expressed the transformed plant cells of IPT gene and will be accumulated phytokinin and grow up to the unusual branch that can not grow root.

The 0.4kbP-DNA fragment of describing among the embodiment 1 is inserted among the pSIM100-OD-IPT to produce pSIM111 (Fig. 1; SEQ ID NO.:4).

In order to check the conversion plant of using pSIM111 whether can obtain to contain P-DNAs (comprising any sequence between the P-DNA end) and not having other carrier main chain, the expression casette of neomycin phosphotransferase (NPTII) is inserted among the P-DNA of pSIM111 and produces pSIM108 (Fig. 1).

By pSIM108 relatively with contain transformation frequency between the control vector of the modification P-DNA with conventional T-DNA edge sequence, check P-DNA end is being supported the efficient of DNA in shifting.This control vector, being called pSIM109, is by right with oligonucleotide: whole P-DNA that 5 ' ACTAGTGTTTACCCGCCAATATATCCTGTCAGAG 3 ' (SEQ ID NO.62) and 5 '-AAGCTTTGGCAGGATATATTGTGGTGTAAACGAAG-3 ' (SBQ ID NO.63) amplification contain the NPTII expression casette produce.Second control vector using among these embodiment is conventional two carrier pBI121 (gene pool registration number AF485783), and it contains the identical NPTII expression casette that inserts on the T-DNA of rule.According to following method two carriers are incorporated in the agrobacterium tumefaciens lba4404 cell.There was under the condition of 1 μ g carrier DNA incubation in competence LB4404 cell (50 μ L) 5 minutes at 37 ℃, in liquid nitrogen (-196 ℃ approximately) freezing about 15 seconds, at 37 ℃ of incubations 5 minutes again.After adding 1ml liquid broth culture (LB), treated cell was grown 3 hours at 28 ℃, and was tiled on the LB/ agar that contains Streptomycin sulphate (100mg/L) and kantlex (100mg/L).Then from single LBA4404 clone's overnight culture carrier of separating DNAs and by the restricted enzyme cutting analysis inspection to determine whether to exist complete plasmid DNA.

The LBA4404 ∷ pSIM108 cell growth of the overnight growth by making 10 times of dilutions 5-6 hour, 2, centrifugal 15 minutes sedimentation cells under the 800RPM, with MS liquid nutrient medium (Phytotechnology) the flushing cell that has replenished sucrose (3%, pH 5.7) and in same medium re-suspended cell to OD _600nmBe that 0.2 test of carrying out the model plant tobacco transforms.Then infect the leaf explant of tobacco plant of the growth in vitro in 4 ages in week with suspension.With the tobacco explant that infects at 25 ℃ in Percival growth room (illumination in 16 hours), containing collaborative substratum (the 1/10MS salt of 6g/L agar, 3% sucrose, pH5.7) go up cultivation 2 days, transfer to subsequently in the M401/ nutrient agar that contains Ticarcillin/Clavulanate Acid (150mg/L) and kantlex (100mg/L).Be presented at the callosal quantity that each explant is produced in ensuing 4 weeks in the table 3.Our data show the efficient high about 50% than T-DNAs in the tobacco that transforms by natural terminal or P-DNAs that conventional T-DNA edge sequence is described.The P-DNA transformation efficiency that increases is attributable to other unknown constitutional features of different CG content or P-DNA.

Embodiment 3

Transform potato with the P-DNA carrier

This embodiment shows and can use P-DNA that DNA is transferred to the potato cell from edaphic bacillus in the mode of similar T-DNA.

According to following method by with age in soil bacillus strain infection 4 week growth in vitro the stem explant of RussetRanger plantlet carry out Transformation of potato.The culture growth of the overnight growth of 10 times of dilutions 5-6 hour, 2, centrifugal 15 minutes sedimentation cells under the 800RPM, MS liquid nutrient medium (Phytotechnology) flushing with having replenished sucrose (3%, pH 5.7) is resuspended to OD in same medium _600nmBe 0.2.Follow internode potato sections with resuspended cell infection 0.4-0.6mm.With the stem that infects at 22 ℃ in Percival growth room (illumination in 16 hours), containing collaborative substratum (the 1/10MS salt of 6g/L agar, 3% sucrose, pH 5.7) go up and cultivated 2 days, transfer to subsequently in the corpus callosum inducing culture that contains Ticarcillin/Clavulanate Acid (150mg/L) and kantlex (100mg/L) (CIM has replenished the MS of ribosylzeatin, 0.1mg/L naphthylacetic acid and the 6g/L agar of 3% sucrose 3,2.5mg/L).After cultivating 1 month on the CIM, explant is transferred in the branch inducing culture that contains Ticarcillin/Clavulanate Acid and kantlex (be respectively 150 and 100mg/L) (SIM has replenished the MS substratum of 3% sucrose, 2.5mg/L ribosylzeatin, 0.3mg/L gibberic acid GA3 and 6g/L agar).3-4 counts the quantity of the explant that develops into the transgenosis corpus callosum and/or sprout after week.As shown in the tobacco, the quantity of the callosal stem explant of demonstration that infects with pSIM108 is higher than the quantity (table 3) that those infect with conventional two carrier pBI121 in control experiment.The branch that grows from these corpus callosums can be divided into two different types subsequently.First type branch can't be distinguished on phenotype with the contrast branch that transforms with LBA ∷ pBI121.Second type branch shows the IPT phenotype.The shoot growth that the back is one type is obstructed, and very little leaf is only arranged, and shows bright green extremely yellow color, just can not take root in case transfer in the no hormone culture-medium.For whether the branch of further determining the IPT phenotype contains the IPT gene of stable integration in their genomes, all branches are transferred to contain in the Magenta box that has replenished 3% sucrose and 150mg/L Ticarcillin/Clavulanate Acid MS substratum, allow 3 to 4 weeks of regrowth, be used for DNA isolation.This DNA of plants is as the template in the PCR reaction, and this reaction contains that to be designed to the oligonucleotide that is annealed on the IPT gene right: 5 '-GTCCAA CTT GCA CAG GAA AGA C-3 ' and 5 ' CAT GGA TGA AAT ACT CCT GAGC 3 '.As shown in Table 4, strict dependency between IPT phenotype and the existence of IPT gene is further determined in the PCR experiment.Also check in the plant that from transform, obtains and whether have main chain DNA with pBI121.This finishes by reacting at the enterprising performing PCR of separated DNA from transformation event, " pBI121 main chain primer ": 5 ' CGGTGTAAGTGAACTGCAGTTGCCATG 3 ' (SEQ ID NO.64) and 5 ' CATCGGCCTCACTCATGAGCAGATTGS 3 ' (SEQ ID NO.65) are used in this reaction.The main chain integration has taken place in the bar carrying means that amplifies 0.7kbp.By comparison sheet 4 data presented, deducibility is for P-DNA carrier and T-DNA main chain, and integrating frequency is similar.

Carry out second PCR experiment and whether do not contain any other main chain sequence with the plant of the no IPT of check.Because the IPT expression cassette is near left hand edge sample sequence, thus with the oligonucleotide of this experiment to being designed to be annealed near on the main chain sequence of right hand edge sample sequence: 5 ' CACGCTAAGTGCCGGCCGTCCGAG 3 ' (SEQ ID NO.66) and 5 ' TCCTAATCGACGGCGCACCGGCTG 3 ' (SEQ ID NO.67).The data of this experiment further confirm that plant that the IPT gene is positive also is positive to these other parts of main chain.

Potato mutation RUsset Burbank is similarly tested.According to the evaluation of IPT phenotype, show that the main chain integrating frequency of pSIM108 and pSIM109 can be compared (seeing Table 4 and 5) with among the Russet Ranger those.

Embodiment 4

The Transformation of potato enzyme inhibitor gene

Use conventional method for transformation, this embodiment shows that the new Transformation of potato enzyme inhibitor gene of overexpression will improve the processing and the health characteristic of potato tuber.

Design the new potato homologue (Greiner etc. of following primer with amplification tobacco vacuolus converzyme inhibitor Nt-inhhl, Nature Biotechnology, 17,708-711,1999): 5 ' AAAGTTGAATTCAAATGAGAAATTTATTC 3 ' (SEQ ID NO.68) and 5 ' TTTTAAGCTTTCATAATAACATTCTAAT 3 ' (SEQ ID NO.69).Carry out amplified reaction by mixing following ingredients: 4 μ l DNA of plants, 2 μ l primer (10pM/ml), the anti-phase primer of 2 μ l, 25 μ l Hot Start Master Mix (Qiagen CatalogNr.203443) and 17 μ l water forward.Use PTC-100 thermal cycler (MJ Research) to allow this reaction mixture accept the processing of following polymerase chain reaction (PCR) condition: (1) 95 ℃, 5 minutes (1 circulation), (2) 94 ℃, 1 minute; 45 ℃, 1 minute; And 72 ℃, 4 minutes (35 circulations), (3) 72 ℃, 10 minutes (1 circulation).On 0.8% sepharose, add whole products, use QIAquick gel extraction kit (Qiagen, CA) band of 540 base pairs of purifying from gel.(Promega, (GibcoBRL MD) is transformed among intestinal bacteria (E.coli) the DH5 α WI) to go up and use the efficient competent cell of Max then the fragment of this purifying to be connected to pGEM-T Easy.The sequential analysis of isolating recombinant plasmid dna shows from the DH5 α that transforms, and has the single open reading-frame (ORF) of 543 base pairs formations of 181 amino acid proteins (SEQ ID NO.:5) of being inferred by coding; Bunch contrast shows that with Nt-inhh 70% homology (Fig. 2) is arranged.This high-caliber homology expands to 15 amino acid whose N-terminal districts, shows potato homologue target vacuole.Be enjoyably, Transformation of potato enzyme inhibitors homologue is called St-inhl, with converting enzyme inhibitor (the patent WO98/04722 of the patent tobacco cell wall that is called Nt-inhl; Fig. 2) 43% homology is only arranged.

Although there is the St-inhl gene in the potato tuber of unmodified, its expression level is not enough to all suppress saccharase and reduces cold inductive sweeten.In order to improve the storing property of potato, on the promotor of the new increase stem tuber of particle adhesion starch synthase gene (GBSS), known this promotor promotes the high level expression of gene in the stem tuber with the St-inhl gene fusion.Use the primer 5 ' GAACCATGCATCTCAATC 3 ' (SEQ ID NO.70) and the reverse primer 5 ' GTCAGGATCCCTACCAAGCTACAGATGAAC 3 ' (SEQ IDNO.71) of forward, carry separation GBSS promotor the training kind Russet Ranger from potato by carrying out the PCR reaction.The sequential analysis of the amplified production of cloning in pGEM-T shows that this new promotor contains 658 base pairs (SEQ ID NO.:6).Then promotor/the gene fusion that obtains is connected to 3 ' of potato ubiquitin gene and regulates sequence (UbiT; Thereby guarantee the suitable termination that the saccharase suppressor gene is transcribed SEQ ID NO.:7).

Between the T-DNA edge of two carriers sequence, insert this expression cassette, use the carrier pSIM320 that obtains to transform above-described Russet Ranger.With 9 independently 3 cutting in the transgenic lines be planted in the soil and 4 weeks of growth in (illumination 11 hours, 20 ℃) in the growth room.Then at least 3 tubercles of results and transferring in 4 ℃ the refrigerator from each strain to induce cold sweetening.After 4 weeks, by use Accu-Chek instrument and check bar (RocheDiagnostics, IN) or glucose oxidase/peroxidase reagent (Megazyme Ireland) measures glucose level in the tubercle of these refrigerations.The average glucose level that transform with these levels and 6 unconverted strains with the carrier in pSIM110 source 6 " vehicle Control " that lack the saccharase suppressor genes are compares.As shown in the table 6,3 transgenic lines cumulative glucose is less than " vehicle Control " 40% in being, shows that Transformation of potato enzyme inhibitors homologue has functionally active.

The amount that following experiment shows the reducing sugar that exists in the stem tuber is relevant with the output of acrylamide during stem tuber is processed.The fresh Russet Ranger potato tuber of results and be housed in 4 ℃ and sweeten from the farmland with the slander conduction cooling; The contrast stem tuber storage is at 18 ℃.After 4 weeks, measure the glucose level in the two chunk stems.Subsequently, the flushing stem tuber, Fahrenheit 165 degree bleachings 8 minutes or 12 minutes, be cut into 0.290 * 0.290 little, spend immersion 1% sodium pyrophosphate solution in Fahrenheit 160,, spend fried 40 seconds to reach 64 ± 2% of initial fried water content in Fahrenheit 390 at the dry 14+2% of Fahrenheit 160 degree up to the forfeiture dry weight, spend freezing 20 minutes in Fahrenheit-15, in initial 6 minutes, shake pallet 2-3 time.Then analyze the levels of acrylamide of the French fried food product that obtains by Covance laboratory (WI).As shown in table 7, the level of glucose is lower than the level that detects of 0.1mg/g in the stem tuber of 18 ℃ of storages, and the stem tuber of cold storage contains the glucose of average 3.4mg/g.This table is also shown in the levels of acrylamide that contains in the fried food product of being produced by latter potato and exceeds about 10 times than the fried food product of being produced by the potato that is housed in 18 ℃.Even, still in the acrylamide accumulation, obtain 5 times difference (table 7) to using the potato of 18 ℃ of storages than the fried food product of producing similar color (be respectively 78 and 71 color ids) at the also short bleaching time of the potato of 4 ℃ of storages.Therefore, as if in stem tuber, between the accumulation of acrylamide direct contact is arranged in the fried food product in the amount of reducing sugar such as glucose and these stem tubers source.

Whether limit the accumulation of processing inductive acrylamide for the level of measuring reduction glucose in the pSIM320 strain, pSIM320 strain tubercle wedging thing with the cold storage of processing, bleached 8 minutes, in 0.5%SAPP, soaked 30 seconds, spend dry 4.5 minutes in Fahrenheit 160, fried 40 seconds of Fahrenheit 380 degree, freezing 15 minutes of Fahrenheit-15 degree, in Fahrenheit 160 degree dry 3 minutes and 10 seconds at last.Then finished material delivery is carried out acrylamide measures to the Covance laboratory.As shown in table 6, the acrylamide of the French fried food product accumulation minimum level that from tubercle, obtains with minimum quantity glucose.Glucose level reduces 40% in " 320-2 " and " 320-4 " strain, and associated levels of acrylamide reduces 5 times.

Embodiment 5

Leading and tailer sequence with potato R1 gene-correlation

Use conventional method for transformation, this embodiment shows processing and the health characteristic of new leader sequence to improve potato tuber effectively that can use with potato R1 gene-correlation.Also can foretell and to utilize the new tailer sequence relevant in an identical manner with this homologous genes.

As the replacement method of overexpression converting enzyme inhibitor gene, the method that development does not use any actual gene sequence restriction acrylamide to produce.Such inhibition method is based on the R1 gene silencing that stem tuber is expressed.The antisense expression that had shown the 1.9kb gene fragment by deriving from this gene in the past can make the gene silencing relevant with this starch (Kossmann etc., United States Patent (USP) 6,207,880).Yet the antisense expression of big dna fragmentation is bad, because such fragment contains new open reading-frame (ORF) (table 1).As a kind of safer mode of aforesaid method, from potato, separate little leader sequence with the R1 gene-correlation.On whole RNA of Russet Ranger potato plant stem tuber, carry out the rapid amplifying of cDNA end to obtain this leader sequence by 5 ' the RACE test kit that uses GIBCO BRL to provide.Sequential analysis shows that the leader sequence relevant with R1 constitutes (SEQ ID NO.:8) by 179 base pairs.The justice of this leader sequence that will be separated by potato ubiquitin intron (SEQ ID NO.:9) and antisense copy place between GBSS promotor and the UbiT.The expression cassette (SEQ ID NO.:10) that in Fig. 3, shows the leader sequence relevant that obtains with R1.At (Fig. 3; SEQ ID Nos.:12) show similar expression cassette in, it contains from the transcribed spacer of GBSS promotor (SEQ ID NO.:11), replaces the Ubi intron to separate the justice and the antisense copy of R1 tailer sequence.The other variant (SEQ ID NOs.:14-15) that in Fig. 3, shows the more microscler formula that contains the GBSS promotor.

In order to check the leader sequence relevant, will in Fig. 3, insert between the T-DNA edge of two carriers sequence as the KpnI-XbaI fragment by shown expression cassette in the aborning efficient of restriction acrylamide with R1.The edaphic bacillus LBA4404 strain that use has the pSIM332 carrier that obtains transforms Russet Ranger potato.In order to induce the formation of stem tuber, transfer to the branch of 25 independent transformation events of representative in the soil and place (illumination 11 hours, 25 ℃) in the growth room.After 3 weeks, at least 3 tubercle/product are tied up to 4 ℃ of 4 weeks of storage to induce the starch transfer.Measure glucose level in the tubercle of these refrigerations according to the method for describing among the embodiment 4 subsequently, and compare with the average glucose level in unconverted plant and the vehicle Control.As shown in table 8, reduced from glucose level after the tubercle demonstration refrigeration of all 25 strains.Compared with the control, the level of estimating acrylamide in from the French fried food product that shows the tubercle that the R1 expression level reduces has approximately reduced 2 times.Expectation is stronger to the influence of downward modulation R1 genetic expression in sophisticated stem tuber.

As the replacement method based on the leader sequence method, generation contains the justice of the tailer sequence relevant with R1 and the expression cassette of antisense copy.Obtain this tailer sequence by carrying out reverse transcription polymerase chain reaction (RT-PCR) at isolating whole RNA from the microtubers of potato cultivar RussetRanger.Use Omniscript RT test kit (Qiagen, CA) produce complementary DNA, (the anti-phase primer R1-1 of gene specific (5 ' GTTCAGACAAGACCACAGATGTGA 3 ') is used in this reaction for Qiagen, the template of PCR reaction CA) as using Hots tart archaeal dna polymerase then.The sequential analysis of being cloned into the dna fragmentation of the amplification on the pGEM-T shows that the tailer sequence relevant with R1 constitutes (SEQID NO.:16) by 333 base pairs.By the Ubi intron that between GBSS promotor and Ubi terminator, inserts or GBSS transcribed spacer the justice and the antisense copy of tailer sequence separated (Fig. 3; SEQ IDNos.:17-18).In Fig. 3, show the similar type (SEQID NOs.:19-20) that contains big GBSS promotor.

Can measure the level of glucose and acrylamide according to above-described method.The desired display glucose concn reduce about 50% or more stem tuber in frying course also accumulation be less than about 50% acrylamide.Can in the stem tuber of sophisticated farmland growth, further determine to modify health and the storing property of plant through improving.

Can measure the phosphoric acid level in the potato tuber by using the phosphorus (all) (the international formal analytical procedure of 45.1.33AOAC, the 17th edition) in AOAC method 995.11 foods.Prepare sample with acid digestion subsequently by dry ashing in retort furnace.With the dissolved sample, handle and compare then with a series of phosphorus standards (through similar processing) with molybdic acid-ascorbic acid solution.Use dual beam spectrophotometer to carry out colorimetric analysis in 823 nanometers.The expection phosphorus content can significantly reduce, and this is favourable to environment.

Embodiment 6

With the relevant leader sequence of L-alpha-glucan phosphorylase gene

Use conventional method for transformation, this embodiment shows can use new leader sequence processing and the health characteristic effectively to improve potato tuber relevant with potato L-alpha-glucan phosphorylase gene.

Shown in the past and can reduce cold inductive sweeten (Kawchuk etc., United States Patent (USP) 5,998,701,1999) by the segmental antisense expression of the 0.9kb of alpha-glucan phosphorylase gene.Yet the antisense expression of the dna fragmentation that these are big relatively is bad, because they contain new and open reading-frame (ORF) that be not described, just may influence the nutritional quality of food (table 1) if these open reading-frame (ORF)s are expressed in transgenic plant.

As the safer mode of aforesaid method, from the RNA of ripe stem tuber, separate little leading and tailer sequence with L type glucosan phosphorylase gene-correlation.Be used for the employed primer of this purpose to being: 5 '-GGATCCGAGTGTGGGTAAGTAATTAAG-3 ' (SEQ ID NO.72) and 5 '-GAATTCTGTGCTCTCTATGCAAATCTAGC-3 ' (SEQ ID NO.73).Amplification obtains the leader sequence of 273 base pairs, is shown as SEQ ID NO.:21.Similarly, use increase " tailer sequence " (SEQ ID NO.:22) with 158 base pairs of L type glucosan phosphorylase gene-correlation of " directly " primer 5 '-GGAACATTGAAGCTGTGG-3 ' (SEQ ID NO.74) and widow-dT primer.

Use then these trail or leader sequence design expression cassette modifying L type glucosan phosphorylase expression of gene, and so shift the levels of acrylamide that reduces in the fried product by restriction starch.With with embodiment 5 in the similar fashion described make up these expression cassettes, and be described in (SEQ ID Nos.:23-26) among Fig. 3.Use contains the soil bacillus strain infection potato haulm of the two carriers with this expression cassette that are called pSIM216, produces 25 transfer-gen plants.To preserve for 4 weeks to induce cold sweetening at 4 ℃ from the tubercle of these plant.Analyze glucose level then through the refrigeration tubercle.As shown in table 9, the tubercle of all transgenic strains shows that glucose level reduces.

Use demonstration glucose concn reduces by 4 strains (216-2,216-5,216-10 and 216-21 strain) of 50% at least to estimate processing inductive levels of acrylamide.Although levels of acrylamide is similar with contrast in from the fried stem tuber of preceding 3 strains, only accumulate 45% the levels of acrylamide (per 1,000,000,000/136:305) that is equivalent to wild-type from the French fried food product of 216-21 strain.These results further confirm the experiment to overexpression Transformation of potato enzyme inhibitor gene stem tuber of description in embodiment 4, because need the relative big reduction of glucose (with fructose) concentration with thermoinducible acrylamide accumulation in the tubercle that is limited in refrigeration.Because it is more effective to be desirably in the silence of phosphorylase gene in sophisticated " 216 " stem tuber, so it is more remarkable also to be desirably in the reduction of levels of acrylamide in the French fried food product of producing with such stem tuber.Can determine further in sophisticated stem tuber that the plant of modifying has the health and the storing property of improvement.

Embodiment 7

The polyphenol oxidase genes of modifying

Use conventional method for transformation, this embodiment shows that the polyphenol oxidase genes that can use the modification that lacks functional copper binding site is to reduce the scar susceptibility of stem tuber effectively.

Showed in the past, can reduce blackspot scar susceptibility (Steffens, United States Patent (USP) 6,160,204,2000) by the antisense expression of 1.8kb PPO gene.Yet the expression of the reverse complementary sequence of this big gene is not wished to want, because it contains new and open reading-frame (ORF) that do not describe, its coding may influence the nutritional quality (table 1) of food potentially by surpassing 100 peptides that amino acid constitutes.As the safer mode of aforesaid method, modify the PPO gene with coding NOT-function albumen.

From Russet Ranger, separate the potato PPO gene of wild-type by using polymerase chain reaction (PCR) method.At first, isolation of genomic DNA from Russet Ranger bud.Use archaeal dna polymerase and Oligonucleolide primers then: 5 ' CGAATTCATGGCAAGCTTGTGCAATAG3 ' is (SEQ ID NO.75) and 5 ' CGAATTCTTAACAATCTGCAAGACTGATCG 3 ' (PPO-R) (SEQ ID NO.76) the potato PPO gene that increases from potato gene group DNA (PPO-F).Be designed to 5 ' of potato PPO gene-and 3 '-end these complementary.The 1.6kb fragment cloning of amplification to pGEM-TEASY carrier (Promega), is confirmed further that by sequential analysis it represents functional PPO gene (SEQ ID NO.:27).

By this albumen and sweet potato PPO albumen are compared, identify the structural domain among the potato PPO in conjunction with copper, wherein the PPO albumen of sweet potato be presented at 92 contain contain on conservative halfcystine (Cys) residue, 236 glutamine (Glu) residue and with 88 of two avtive spot cupric coordinations, 109,118,240, contain Histidine (His) residue (Klabunde etc. on 244 and 274, Nature Structural Biol., 5:1084-1090,1998).In potato PPO, also there are these halfcystines, glutamine and Histidine.

Produce the PPO gene of non-activity by the method for using the PCR sudden change to replace.Use 3 pairs of primers and wild-type Russet Ranger PPO as template, amplify 3 fragments by Proof Start Taq archaeal dna polymerase (Qiagen).First pair of primer is called P1-F and P2-R, and its sequence is: 5 ' GAGAGATCTTGATAAGACACAACC 3 ' (SEQ ID NO.77) and 5 ' CATTACC ¹ATAAGCC ²CAC ³(1: " A " is mutated into " C " to TGTATATTAGCTTGTTGC 3 ' (SEQ ID NO.78), causes the halfcystine on 186 to be replaced by glycine; 2: " A " is mutated into " C ", causes the halfcystine on 183 to be replaced by tryptophane; 3: " A " is mutated into " C ", causes the Histidine on 182 to be replaced by glutamine).Second pair of primer is called P3-F and P4-R, and its sequence is: 5 ' GTGCTTATAGAATTGGTGGC3 ' (SEQ ID NO.79) and 5 ' TAGTTCCCGGGAGTTCAGTG 3 ' (SEQ ID NO.80).The 3rd pair of primer is called P5-F and P6-R, and its sequence is: 5 ' CTCCCGGGAACTATAGG ⁴AAACATTCCTCT ⁵(4: " A " is mutated into " G ", causes the glutamine on 326 to be replaced by glycine for CGGTCCTGTCCACATCTGGTC 3 ' (SEQID NO.81) and 5 ' GTGTGATATCTGTTCTTTTCC 3 ' (SEQ ID NO.82); 5: " A " is mutated into " T ", causes the Histidine on 330 to be replaced by leucine).

Use the fragment of 80 base pairs of primer P1-F and P2-R amplification, and digest with BglII.This fragment contains a sticky end (BglII) and a passivity end, and has 3 sudden changes on copper binding site I.Use primer P3-F and P4-R amplification and contain the terminal and sticky end (XmaI) of a passivity with the 0.4kb fragment that XmaI digests.Use primer P5-F and P6-R amplification 0.2kb fragment, and digest with XmaI and EcoRV.This 3rd fragment with sticky end (XmaI) and passivity end (EcoRY) has 2 sudden changes in copper binding site II.The BglII and the EcoRV fragment that then replace clone's wild-type potato PPO with the pcr amplified fragment of top 3 connections.There are whole 5 point mutation (SEQ ID NO.:28) in the PPO gene of further determining to modify by sequential analysis.Expression cassette for the PPO (mPPO) that produces modification, following 4 fragments are coupled together simultaneously: (1) contains the BamHI-HindIII fragment of GBSS promotor, (2) contain the HindIII-SacI fragment of mutant PPO, (3) contain the SacI-KpnI fragment of Ubi-3 terminator, and (4) are with the plasmid pBluescript of KpnI and BamHI digestion.Between the edge of two carriers sequence, insert this expression cassette then and produce pSIM314.

Estimate the efficient of mPPO expression casette by transform Russet Ranger stem explant with pSIM314.The joint cutting that will contain the transgenic plant of this expression cassette places the MS substratum that has replenished 7% sucrose., separate microtubers and identify the PPO activity after 5 weeks 18 ℃ of dark culturing.Purpose is pulverized the 1g potato tuber in liquid nitrogen hereto.Then this powder is added to 5ml and contains in 50mM MOPS (3-(the N-morpholine subbase) propanesulfonic acid) damping fluid (pH 6.5) of 50mM catechol, at room temperature rotating and culturing is about 1 hour.The precipitated solid part is transferred to supernatant liquor in another test tube then, measures the PPO activity by the variation of measuring 410 pairs of times of OD.As shown in table 10, compare with unconverted contrast or with the contrast that the construction that does not contain mutant PPO gene transforms, isolating microtubers shows that polyphenol oxidase activity significantly reduces from some transgenic lines.Observing the intensive of PPO activity in " 314-9 ", " 314-17 " and " 314-29 " strain reduces.In order to check mutant PPO expression of gene whether also to reduce the PPO activity in the tubercle, in soil, plant the plantlet of taking root of transgenic lines, and in growth 4 weeks of indoor cultivation.The PPO of isolating tubercle measured show, the PPO that in most of examples of determining, reduces in the microtubers active with tubercle in the activity relevant (table 10) that reduces.Can breed the transgenic lines that shows the active reduction of PPO, in greenhouse and farmland, test further to determine " low scar " phenotype in the sophisticated stem tuber.Because microtubers and tubercle are expressed multiple polyphenoloxidase, so wherein some and the only limited sequence homology of target polyphenoloxidase of mainly in sophisticated stem tuber, expressing, as the ripe stem tuber of " 314-9 " and " 314-17 " strain in can expect PPO activity even can more extremely reduce.Data show that the overexpression of the active PPO gene of idle energy can cause reducing the scar susceptibility.Also can in the ripe stem tuber of farmland growth, further confirm the health and the storing property through improving of modified plant.

Embodiment 8

Tailer sequence to the special polyphenol oxidase genes of the non-face tissue of potato tuber

Use conventional method for transformation, this embodiment shows, can use new tailer sequence with potato PPO gene-correlation to reduce the scar susceptibility of stem tuber effectively.

The tailer sequence of the PPO gene-correlation that uses reverse transcription PCR also to separate and in potato tuber, express.The primer of first PCR reaction is PPO-1 (5 ' GAATGAGCTTGACAAGGCGGAG 3 ' (SEQ ID NO.83)) and widow-dT; The primer of second nested PCR reaction is PPO-2 (5 ' CTGGCGATAACGGAACTGTTG 3 ' (SEQID NO.84)) and widow-dT.The sequential analysis of being cloned into the dna fragmentation of the amplification on the pGEM-T shows the tailer sequence (SEQ ID NO.:29) that has 154 base pairs.Then the justice of this tailer sequence that will be separated by the Ubi intron and antisense copy are fused on above-described GBSS promotor and the Ubi3 terminator, to be created in the expression cassette shown in Fig. 3 (SEQID NO.:30).In Fig. 3, show and contain the segmental selectable construction of the tailer sequence that is separated by the GBSS transcribed spacer (SEQ ID NO.:31).In Fig. 3, show similar type (SEQ ID Nos.:32-33) with big GBSS promotor.Be that mainly the tailer sequence (representing with P-PPO3 among Fig. 4) of the PPO gene of expressing in ripe stem tuber is different from the tailer sequence (representing with PPOM-41 and PPOM-44 among Fig. 4) of the PPO gene of expressing in other tissue that is mainly comprising microtubers enjoyably.Because therefore the low homology between the tailer sequence different and the PPO gene-correlation uses the P-PPO3 tailer sequence will only cause the silence of the special PPO gene of sophisticated stem tuber.Owing to be difficult to finish the silence of this very specific gene, thereby shown that the use non-coding sequence carries out the advantage of gene silencing with the sequence of PPO gene itself.In order to show the active degree of PPO, the catechol of 0.5ml 50mM is dropped in transfer pipet on the tangent plane of tubercle of the genetic modification of thinly slicing.Compared with the control, the visible brown stain of stem tuber district reduces about 5 to 10 times.Be, on the potato crust, not observe brown stain enjoyably although be like this.As if the tailer sequence that uses makes the main PPO gene silencing of expressing but not expressing specifically in epidermis in cortex and marrow.Because the PPO gene may play certain effect in certain defensive raction, be useful so this unexpected discovery may not be subjected to some pathogenic agent to attempt to infect by crust to protection stem tuber itself.For quantitative assay PPO activity, measure according to the method for describing among the embodiment 7.Table 11 shows to be compared with unconverted contrast, and active reduction of PPO is up to 80% in the tubercle of conversion.Because compare with microtubers with tubercle, these stem tubers are more mainly expressed target PPO gene, so estimate the level reduce even will be more in ripe stem tuber.Can in ripe stem tuber, further confirm characteristic as the improvement of " 217-7 " and " 217-26 " strain.

Embodiment 9

Improve the expression cassette of Resistant starch level

The ratio that increases amylose starch/amylopectin in the stem tuber can further improve the nutritive value of potato product.A method that can improve amylose content is based on the antisense expression (Schwall etc., Nature Biotechnology18:551-554,2000) of coding Q-enzyme (SBE) I and II gene.The shortcoming of this method is that (1) is by utilizing simultaneously reticent two the heterogeneic efficient of antisense technology very low, (2) antisense expression of big relatively SBE-I and SBE-II gene order causes undesirable expression of open reading-frame (ORF) (table 1), (3) the corresponding construction that comprises two antisense expression boxes is unnecessary big and complicated, thereby, increase the chance of reorganization and reduced transformation frequency.

The method of amylose content is based on the expression of the tailer sequence all relevant with two genes in our the raising potato.These tailer sequences (SEQ ID NO.:34 and 35) are isolating to carrying out with following primer: 5 ' GTCCATGATGTCTTCAGGGTGGTA 3 ' (SEQ IDNO.85), 5 ' CTAATATTTGATATATGTGATTGT 3 ' (SEQ ID NO.86), 5 ' ACGAACTTGTGATCGCGTTGAAAG 3 ' (SEQ ID NO.87), and 5 ' ACTAAGCAAAACCTGCTGAAGCCC 3 ' (SEQ ID NO.88).Single promoters driven is separated by ubiquitin-7 intron, is thereafter the justice of two tailer sequences of ubiquitin-3 terminator and the expression that antisense merges.All the size of expression cassette only is 2.5kb.

Embodiment 10

Develop unmarked method for transformation

This embodiment shows that the stable integration that does not need the selected marker also can transform plant effectively.

This method at first utilizes the nuclear DNAs of targeted plants often can not be incorporated into phenomenon in the genome of vegetable cell subsequently.The inventor has obtained wonderful discovery, places last 5 day of plant culture of containing suitable selectivity reagent by the explant that will infect, and may select the cell that temporary transient expression contains selected marker's nonconformity T-DNA.Second phenomenon that is used to develop current method is the identical plant nucleolus of the frequent target of T-DNAs of different two carriers.By using two different two carriers: a selective marker that contains on the T-DNA, another contains T-DNA or the P-DNA with actual interest sequence, can use of short duration selective system and obtain a large amount of corpus callosums, branch or plant, its important part is represented unmarked transformation event.

Use is called as conventional two carriers of pSIM011 and represents the carrier with " interest sequence ", and in this test case, the interest sequence is the expression casette that is positioned at the β glucuronidase (GUS) on the conventional T-DNA.Second the two carrier that uses in these experiments contain the expression cassette that comprises by the terminator sequence of neomycin phosphotransferase (NPTII) gene of the strong promoter of ubiquitin-7 gene driving and nopaline synthase (no) gene thereafter between the T-DNA edge of pSIM011 derivative sequence.

Surprisingly, also can replace the no terminator by terminator (gene pool registration number V01292, SEQ ID NO.56) and obtain high-caliber of short duration NPTII gene expression dose with Alcohol Dehydrogenase from Yeast 1 (ADH) gene.This discovery is very interesting, because yeast ADH1 terminator and any plant terminators all do not have homology.Importantly be to should be noted that here many yeast terminators can not play one's part to the full in plant.For example, behind the gus gene of Miao Shuing be in the above yeast different-the similar experiment of 1-cytochrome c (CYC1) terminator (gene pool registration number SCCYT1) in, almost do not observe gus gene and express.Produce carrier by replace the no terminator with yeast ADH1 terminator through improving with selected marker NPTII.The two carriers that contain the selected marker who is useful on of short duration conversion are called as " LifeSupport " (Fig. 5).

With two agrobacterium tumefaciens lba4404 strain while potato-infecting stem explants that contain pSIM011 and LifeSupport respectively.The culture of the overnight growth of each bacterial strain of 1/10 dilution was grown 5-6 hour before their precipitations, washed and be resuspended to OD according to the method for describing among the embodiment 3 _600nmBe 0.4.Then in order to each bacterium 0.2 (OD _600nm) the re-suspended cell of whole density infect 0.4-0.6cm internode potato fragment.Handle the stem that infects as the method in embodiment 3, the key distinction is: the selection of carrying out with kantlex is limited to preceding 5 days that cultivate in the corpus callosum inducing culture.Then, allow explant further to grow among antibiotic fresh CIM of non-selectivity and the SIM only containing Ticarcillin/Clavulanate Acid (timentine) 150mg/L.To the blade of the branch that grows from infect the corpus callosum of growing in days about 3 months from the stem that 40-60% infects, check GUS expresses the performing PCR analysis of going forward side by side and contains with evaluation and be interested in sequence but do not contain the incident of marker gene.As shown in table 12,11% branch is represented unmarked transformation event.

Also use two above-mentioned bacterial strain method transformation of tobacco.The branch of growing in about 2 months is carried out GUS to be measured and pcr analysis.The high frequency (18%) of the unmarked transformation event that process is identified means that the method for being developed can be used for non-potato plants kind (table 12).

Importantly be, infect in proper order rather than infect simultaneously, caused improving the efficient of unmarked conversion with two different edaphic bacillus bacterial strains.By with containing the soil bacillus strain infection potato haulm explant of pSIM011, the explant that infects is placed collaborative culture plate last 4 hour, infect again with the LifeSupport carrier then, found the wonderful effect of infection in proper order.In this embodiment, method is handled the explant of double infection as described previously.As shown in table 13, between two different infection, cause 4 hours retardation time the frequency of unmarked transformation event in the potato to increase by 2 times.

Embodiment 11

Carry out precise breeding with DSIM340

This embodiment shows the efficient of precise breeding.Be inserted in the potato by genetic elements and (see embodiment 1,4 and 7), use unmarked conversion (embodiment 10) to improve the health and the agronomic characteristics of potato plant potato.

Be inserted among two carrier pSIM112 ' by expression cassette mutant PPO and converting enzyme inhibitor, generation contains the converting enzyme inhibitor that inserts and two carriers of two expression cassettes of mutant polyphenol oxidase genes between the P-DNA end, be called pSIM340 (Fig. 1).With pSIM340 and further improved LifeSupport carrier while potato-infecting stem explant.Then the explant of coculture infection carries out of short duration selection, and induces breeding and produce the branch that discussed the front.After 3 months, transfer to small stems in the new substratum and allow 3 weeks of its regrowth.According to the method for describing in embodiment 2 and 3, branch is carried out phenotype analytical then, collect blade material and be used for the P-DNA of analysis of molecules to determine whether to have main chain, marker gene and have the interest sequence.As shown in table 14,1.2% incident is represented the plant that contains the modification P-DNA of pSIM340 but do not have LifeSupport.The frequency of this unmarked conversion is lower than the frequency of using T-DNA to transform, and this shows to exist between P-DNA and T-DNA once more difference on the function.

Embodiment 12

Select the stable integration of anti-LifeSupport T-DNAs

This embodiment shows, selects the stable integration of anti-LifeSupport T-DNAs can improve the efficient of precise breeding method by the cytosine deaminase gene that uses bacterium.

The embodiment of front shows, uses the efficiency ratio of the unmarked conversion of the P-DNA that modifies to use being several times lower than of conventional T-DNA.Only contain the efficient of modifying the P-DNA branch in order to improve generation, between the T-DNA edge of LifeSupport carrier sequence, insert and comprise bacterium Isocytosine deaminase (codA) and uracil phosphoribosyl transferase (upp) gene (InvivoGen, CA) expression cassette that suicide gene merges produces pSIM346 (Fig. 5).Contain the stem explant of the strain infection potato of pSIM346 with a bacterial strain that contains pSIM340 with another, with being placed in the following substratum: in (1) collaborative substratum 2 days, (2) in the CIMTK substratum to select of short duration marker gene to express 5 days, (3) in the CIMT substratum with the vegetable cell breeding of the marker gene that allows transient expression 30 days, (4) contain in the SIMT substratum of the nontoxic 5-flurocytosine of 500mg/L (5-FC), wherein nontoxic 5-flurocytosine is expressed codA:upp by vegetable cell and is changed into deleterious 5 FU 5 fluorouracil (5-FU), to select the stable integration of anti-LifeSupport T-DNA.Corpus callosum produces branch in SIMT in 4 weeks.These branches are transferred in the MS substratum that contains Ticarcillin/Clavulanate Acid (timentine), and allowed growth up to obtaining enough tissues that is used for pcr analysis.From 100 branches, extract DNA then and be used to determine whether to exist P-DNA, LifeSupport and main chain.As shown in Table 15, none contains LifeSupport T-DNA in the analyzed branch, shows for the first time, can be with the codA:upp gene fusion as the preceding negative selected marker of regeneration.More importantly be that our result shows that the feminine gender selection that anti-LifeSupport T-DNA integrates improves the frequency of the branch of the P-DNA that only contains modification.Select to combine with the feminine gender of the stable integration of anti-codA:upp gene fusion by the positive selection that of short duration marker gene is expressed, only contain the frequency ratio of modifying the P-DNA branch and only use the positive of of short duration marker gene expression to select about 5 times (table 15) of height.

By using LifeSupport carrier pSIM350 (Fig. 5), the efficient of unmarked conversion even obtain extremely increasing, this carrier and pSIM346 are similar but contain the codA gene of replacement codA:upp gene fusion.Method is handled the stem explant of the potato that infects simultaneously with pSIM340 and pSIM350 as described above, checks resulting 51 branches whether the incident in the T-DNA district of pSIM340 takes place only to contain with the method for molecules.Be that this pcr analysis shows that some branches contain codA gene (table 15) enjoyably.This discovery shows that codA and codA:upp are different in plant, is not negative selection marker closely.More importantly be to show the incident of the unmarked conversion of a large amount of branches (29%) representative.

Not to infect explant with pSIM340 and pSIM350 simultaneously, can further improve efficient but sequentially infect.By infecting explant with pSIM340 and infecting again with pSIM350 after 4 hours, expect that the frequency of unmarked conversion will be about 30-40%.

Embodiment 13

Reduce the integration of LifeSupport T-DNAs

This embodiment shows, reduces LifeSupportT-DNA by the virD2 gene that uses the Ω sudden change and is incorporated in the Plant Genome, can improve the efficient of precise breeding method.

The Ω zone that always shows edaphic bacillus albumen vird2 is incorporated into ability very important (Mysore etc., Mol Plant MicrobeInteract 11:668-83,1998) in the Plant Genome for this albumen support with T-DNA.Observe based on this, produced the LifeSupport carrier that contains the modification of the virD2 protein expression box of the sudden change of the Ω on the SacII site in the main chain sequence that is inserted into them.Obtain this expression cassette by amplification 2.2kb dna fragmentation from plasmid pCS45 (Walt doctor Ream gives, Oregon university, OR, USA-, SEQ ID NO.:36).Use is called the LifeSupport derivative that contains this expression cassette of pSIM401 Ω (Fig. 5), supports the conversion of the P-DNA that passes through modification of usefulness pSIM340 to potato plant.After of short duration selection and branch are induced, whether there is transgenosis with 100 branches of molecules method check.As shown in table 15,4.4% branch only contains the P-DNA of modification, and this shows that use Ω-virD2 has improved about 4 times (table 15) with the efficient that meta-tag transforms.

Being increased to 8.1kb by the size with LifeSupport T-DNA from 3.7kb (among pIM401 Ω) (is being called as in the pSIM401 Ω derivative of pSIM341 Ω; Fig. 5) further raise the efficiency.By the branch of from the potato haulm explant that infects simultaneously with pSIM340 and pSIM341 Ω, regenerating, show the incident (table 15) of the unmarked conversion of 7 (7%) representative in 81 analysis incidents.

By infecting explant with pSIM340 and LifeSupport in proper order rather than infecting simultaneously, can obtain further raising.With with embodiment 10 in the similar fashion described, by infecting explant, infect again with LifeSupport after 4 hours with pSIM340, the frequency that only contains the P-DNA plant of modification can increase about one times.

Embodiment 14

The method of 1 bacterial strain is used in exploitation

This embodiment shows, also can obtain high-frequency unmarked conversion by the single edaphic bacillus bacterial strain that use contains P-DNA carrier and LifeSupport.

Two compatible two carriers that generation can be kept in edaphic bacillus simultaneously.Use this system to be used for only integrating the P-DNA of modification, rather than be used for the T-DNAs (Komari etc., United States Patent (USP) 5731179,1998) that two of stable integrations contain interesting DNA and marker gene respectively.

First carrier is called as pSIM356, contains comprising by the expression cassette of Ubi7 promoter-driven GUS gene and UbiT subsequently of inserting between the P-DNA end.The IPT expression of gene box (Fig. 1) that the main chain of this carrier partly contains bacterium replication orgin from pVS1 and pBR322, be used for spectinomycin resistance gene that bacterium selects and can select anti-main chain to integrate plant.Second carrier is called as pSIM363, contains comprising by the expression cassette (Fig. 5) of the NPTII gene of Ubi7 promoters driven and yeast ADH1 terminator subsequently of inserting between conventional T-DNA edge sequence.The main chain of this carrier partly contains the bacterium replication origin of ColE1 (gene pool V00268) and oriV (gene pool M20134), and is used for the kalamycin resistance gene that bacterium is selected.

Check uses pSIM356 and pSIM363 to improve the thought of unmarked transformation frequency in 100 tobacco branches.As shown in table 16, about 19% regeneration branch contains the interest DNA of marker-free.Be used for potato by method, find also can improve the efficient of unmarked conversion 1 bacterial strain of this use.9 (15%) in 60 independent branches of check contain pSIM340T-DNA and lack LifeSupportT-DNA (table 16).

Can be with the method associating described among the method for using 1 bacterial strain and the embodiment 12 so that the positive that of short duration marker gene is expressed select to combine with the feminine gender selection of anti-codA stable gene integration.For this purpose, developed LifeSupport carrier pSIM36 (Fig. 5).Can use the edaphic bacillus bacterial strain that contains this carrier and P-DNA carrier together to produce effectively only to contain and be positioned at the plant of stable integration at the interest genes expression cassette of their genomic P-DNA.

Embodiment 15

Rely on native labelled precise breeding method

Except transforming farm crop to introduce the useful characteristic with only containing the P-DNAs that expects sequence, the present invention also provides the method that transforms this class plant with the P-DNAs that contains other natural marker gene.New and the natural marker gene that we select is Arabidopis thaliana (Arabidopsis) vacuole Na ⁺/ H ⁺The potato homologue of reverse transport protein gene and clover alfin-1 gene.These expression of gene not only allow to identify transformation event, and salt tolerance are provided for the conversion plant.Therefore in the farmland that area increases gradually, the high salinity level will influence the potato plant that contains the salt tolerance mark lessly.

The use oligonucleotide is right: 5 ' CCCGGGATGGCTTCTGTGCTGGCT 3 ' (SEQ IDNO.89) and 5 ' GGTACCTCATGGACCCTGTTCCGT 3 ' (SEQ ID NO.90), the vacuole Na of two kinds of forms of amplification in the cDNA of the late blight resistant variety that is called " LBR4 " that obtains from U.S. potato gene storehouse (WI) ⁺/ H ⁺The reverse transport protein homologue is called Ps t (potato salt tolerance).Their sequence is shown as SEQ ID NO.:37 and 38.Use primer 5 '-CCCGGGTATGGAAAATTCGGTACCCAGGACTG 3 ' (SEQ ID NO.91) and 5 '-ACTAGTTAAACTCTAGCTCTCTTGC 3 ' (SEQ ID NO.92) from LBR4 potato DNA, to increase and the 3rd gene of alfin-1 homologous (SEQ ID NO.:39).By the fusion of insertion between the conventional T-DNA edge sequence of the pSIM341 carrier of modifying, estimate the efficient of Pst gene as transformation marker with the Ubi7 promotor.At of short duration selection after date, allow kalamycin resistance cell proliferation and develop into branch.Then these branches are transferred in the substratum that contains 100-150mM sodium-chlor.The branch of salt tolerance is represented the transformation event of the T-DNA of the pSIM341 that contains modification.

Embodiment 16

The specific promotor of stem tuber

The specific promotor of new isolating stem tuber can replace the expression cassette of GBSS promotor to describe among the development embodiment in front of use.By specific primer, use inverse PCR from the genome of Russet Burbank potato plant, to separate this promotor with potato proteinase inhibitor gene (gene pool registration number D17332) (SEQ ID NO.39).Contain the efficient of checking the PIP promotor by two carriers of this promoter-driven GUS gene and NPTII marker gene expression cassette by generation.Use contains similar construction that useful GBSS promotor replaces the PIP promotor in contrast.By with the soil bacillus strain infection stem explant that contains two carriers, cultivated altogether 2 days, and on the CIMTK substratum, selected 2 months and acquisition conversion branch.These branches are transferred in the new substratum to induce the formation root, be planted in the soil then.Growth can detect the GUS expression of stem tuber after 3 months in the greenhouse.

Embodiment 17

The preferred construction and the method for transformation of precise breeding

Except pSIM340, can use other many carriers, improve potato plant by being converted with modification P-DNAs.In this class carrier two kinds contain with except epidermis, the expression cassette (seeing embodiment 8) that the justice of the tailer sequence of the PPO gene-correlation of all expressing in institute's tuberosity tissue and antisense copy.Carrier pSIM370 contains the justice of the another one leader sequence relevant with phosphorylase gene and the expression cassette (seeing embodiment 6) of antisense copy.Carrier pSIM371 contains the 3rd expression cassette (Fig. 1) of potato alfin-1 homologue.

The 3rd selectable carrier, be called pSIM372, contain potato alfin-1 homologue expression cassette and the tailer sequence relevant with PPO, with R1 relevant leader sequence and with Starch phosphorylase the justice of the syzygy of relevant leader sequence and the expression cassette of antisense copy.

For the method for using 1 bacterial strain, preferred LifeSupport carrier is pSIM365.For the method for using 2 bacterial strains, preferred carrier is pSIM367, and it contains NPTII and the expression cassette of codA and the expression cassette (Fig. 5) of another Ω virD2 between T-DNA edge sequence in the plasmid main chain.

With the stem explant of 1 the strain infection potato that contains any one carrier among pSIM365 and the carrier pSIM370,371 and 372, perhaps with any one 2 bacterial strains potato-infecting stem explant sequentially that contains respectively in pSIM366 and the preferred interest carrier.After cultivating altogether 2 days and selecting 5 days momently, explant transferred in the substratum breed/regenerate and remove edaphic bacillus (Agrobacterium).After 30 days, transfer to explant identical once more but now also contain in the substratum of 5-FU to remove the incident that contains LifeSupport T-DNAs.The branch that grows on corpus callosum is subsequently transferred in the regeneration culture medium that may contain 100-200mM salt with screening salt tolerance incident.The branch of permission IPT feminine gender is taken root and is developed into sophisticated plant.Estimate that major part (10%-100%) representative in these plant contains the plant of the unmarked and no main chain of the P-DNA with stable integration interest nucleotide sequence in their genomes.

Table 1. polypeptide that potential expression did not characterize in antisense potato system

Gene (employed segmental size)	Polypeptide by the prediction of the ORFs in the DNA of reverse complemental coding
Gene (employed segmental size)		R1(1.9kb)	MSSTSNVGQD CLAEVTISYQ WVGRVINYNF FLLIHWYTVV EASTGITFQI FPIGIRSEDD RSFYEKADRF AWVT
MSSESTFSKT PNGRATDVGI PTEEGTFPFR YAILRDLAPT ISLVNSSADI A
MSSESTFSKT PNGRATDVGI PTEEGTFPFR YAILRDLAPT ISLVNSSADI A	MSEGVGFKSK ILPSFAWRSA NILGSKHVAK QTFPFLARTE TCERTSGMSG VIRATAPSGI SSSPLTDFAT KIVGFS
GLP(1kb)			VCSPALKADK SKSADGTCVD HSRRLIVVLV LYPGMGTSYA TAFISSPPIQ YLFPSDPVET FP
	MLGSLVLPKS PENRKQAVPN PHFQEQHLVP EKPHFLDCGQ GFSKLPQMHQ
	MLGSLVLPKS PENRKQAVPN PHFQEQHLVP EKPHFLDCGQ GFSKLPQMHQ	MVNFLTQGIV DMETAFGSPK MGGFGKEQFG ACVSRSEMDE SGIGAVMVEQ VCS ICSRHFV LSMQI
	GHTP(0.9kb)		MLEGSMWPWN QESMKRAFLN HHFLMLHLFP AQRPPQAADP VCLKHQHMHC GCLSFQLHLS KLAPGDTPLI SSMFALD
MKLCSSIILS IIKQKQVEIL RACFGFPETK TISVFSSVSW NWHIICKSL
MKLCSSIILS IIKQKQVEIL RACFGFPETK TISVFSSVSW NWHIICKSL		MTKKPDRKDN IMPYNFPGTK FLQPIFRNFF LPSLCDKLLK KSISVPQAIT PCWKVQCGHG IKKA
PPO(1.8kb)			TILKLDLHTF NGHFPTASFW NQSHRNSIFI FQSNILQQFS YRQLESNTGN MISITSMNM RQASITPCKL RLIKLICIHS LVHVQKHIEP YIVPIIIRYF IECQYLLLLI FLLCCP
	MKGKEKPREM NLQFFTTNFV STVAISTMNI SLLFKAKRVK GVFIKFPHST RSQLILGYVL LIRRMSRGAD AEFSHRRELV VRNTIDLIGY RRATTVYYIN TFFYMGSTTR LEIRRWYRCS SR
		MEWALARNRI PFFYCPNSLR TSHGKGYDFH RRKRIQSSTN LYLLNPFFSR QLIS IHSTSC PHWHGGSKKS DLNRVSRNYP CLHRFFDEVC HRSRCEPEYE GCFQ
	SBE A(1.2kb)		MNNITHSPIL IPFLEQLNPF ISNCHMQPIV KANTPILNGN TKCRHSANIF TNGNCIWEKP MNKIVDQHQI HNSIHISCES KVFLVVPSES HR
MKFRYPSPPN PIVTSLIILC NAIPRSINDV DGLSRAIKSY ISLSISQNAI VLSPTRA
		SBE B(2.6kb)	MVNIMTSSSM ATKFPSITVQ CNSVLPWQVT SNFIPFVCVL WVEVEYKYQV TTFKHNNLII IIHAAYYLFS
MAKLVTHEIE VPLSSQGHCE KMDHLVKRNS SINNRRSICQ ARHARIHLFV H
MAKLVTHEIE VPLSSQGHCE KMDHLVKRNS SINNRRSICQ ARHARIHLFV H	MFETKLNSGV VWNDWLTVNI RNSNTPNTKL VLLHHVVRTV PSIEIANNFV FLSSRSPFTI DYATIFPVES KF
MLYTSLYISY LSNSMLLPSW TNLHHSYSLN NLSTYLGLPL PGGNQNQFLP QKQAGQGPAY QKHLRQ

Table 3. transformation efficiency

Two carriers	Corpus callosum/tobacco leaf explant ± SE	Corpus callosum/potato haulm explant ± SE
Two carriers	Corpus callosum/tobacco leaf explant ± SE	Corpus callosum/potato haulm explant ± SE	pBI121	7.8±0.6	0.31±0.10
pSIM108	10.2±0.6	0.59±0.07	pBI121	7.8±0.6	0.31±0.10
pSIM108	10.2±0.6	0.59±0.07	pSIM109	12.8±0.6	0.47±0.05

The main chain that table 4. obtains from Russet Ranger transforms is integrated

Two carriers	Sum	The IPT phenotype	For IPT is the PCR male	For the 0.6kb backbone segments is the PCR male
Two carriers	Sum	The IPT phenotype	For IPT is the PCR male	For the 0.6kb backbone segments is the PCR male	pBI121	98	NA	NA	54(55％)
PSIM108	193	138(71％)	137(71％)	NA	pBI121	98	NA	NA	54(55％)
PSIM108	193	138(71％)	137(71％)	NA	PSIM109	133	82(62％)	80(60％)	NA

NA: can not use

The main chain that table 5. obtains from Russet Burbank transforms is integrated

Two carriers	Sum	The IPT phenotype
Two carriers	Sum	The IPT phenotype	pSIM108	79	49(60％)
pSIM109	72	60(84％)	pSIM108	79	49(60％)

Table 6. levels of acrylamide in the French fried food product of pSIM320 tubercle of refrigeration of hanging oneself

Strain	Glucose mg/g (% of reduction)	Acrylamide (PPB)
Strain	Glucose mg/g (% of reduction)	Acrylamide (PPB)	Unconverted	10.2	469
Vehicle Control	10.2	NA	Unconverted	10.2	469
Vehicle Control	10.2	NA	320-2	5.4(47％)	95
320-4	5.8(43％)	107	320-2	5.4(47％)	95
320-4	5.8(43％)	107	320-7	8.7(14％)	353
320-9	7.4(27％)	137	320-7	8.7(14％)	353
320-9	7.4(27％)	137	320-17	6.0(41％)	506
320-21	8.5(16％)	428	320-17	6.0(41％)	506
320-21	8.5(16％)	428	320-33	6.6(35％)	516

NA: can not obtain

Table 7. is from the levels of acrylamide in the French fried food product of unconverted ripe stem tuber

	In 18 ℃ of storages (color id.*)	In 4 ℃ of storages (color id.*)
	In 18 ℃ of storages (color id.*)	In 4 ℃ of storages (color id.*)	Glucose level	＜0.1mg/g	3.4mg/g
Bleached 8 minutes	53PPB(78)	603PPB(56)	Glucose level	＜0.1mg/g	3.4mg/g
Bleached 8 minutes	53PPB(78)	603PPB(56)	Bleached 12 minutes	28PPB(84)	244PPB(71)

*: value is high more to show that the color of the fried product of finishing is bright more

The glucose level of table 8. in the pSIM332 tubercle of refrigeration

Strain	Glucose mg/g (% of minimizing)
Strain	Glucose mg/g (% of minimizing)	Unconverted contrast	11.6±0.5
Vehicle Control	11.5±0.5	Unconverted contrast	11.6±0.5
Vehicle Control	11.5±0.5	332-1	5.4(53％)
332-2	4.8(58％)	332-1	5.4(53％)
332-2	4.8(58％)	332-4	7.0(39％)
332-5	5.8(50％)	332-4	7.0(39％)
332-5	5.8(50％)	332-6	6.9(40％)
332-7	6.0(48％)	332-6	6.9(40％)
332-7	6.0(48％)	332-8	6.8(41％)
332-9	6.6(43％)	332-8	6.8(41％)
332-9	6.6(43％)	332-10	5.4(53％)
332-11	6.1(47％)	332-10	5.4(53％)
332-11	6.1(47％)	332-12	6.4(44％)
332-13	6.4(44％)	332-12	6.4(44％)
332-13	6.4(44％)	332-15	7.7(33％)
332-16	6.5(43％)	332-15	7.7(33％)
332-16	6.5(43％)	332-17	5.3(54％)
332-18	7.1(38％)	332-17	5.3(54％)
332-18	7.1(38％)	332-21	6.3(46％)
332-22	5.4(53％)	332-21	6.3(46％)
332-22	5.4(53％)	332-23	4.2(63％)
332-31	6.0(48％)	332-23	4.2(63％)
332-31	6.0(48％)	332-34	6.2(48％)
332-35	6.4(44％)	332-34	6.2(48％)
332-35	6.4(44％)	332-39	6.7(41％)
332-40	7.5(35％)	332-39	6.7(41％)
332-40	7.5(35％)	332-41	5.7(50％)

The glucose level of table .9 in the pSIM216 tubercle of refrigeration

Strain	Glucose mg/g (% of minimizing)
Strain	Glucose mg/g (% of minimizing)	Unconverted contrast	11.6±0.5
Vehicle Control	11.5±0.5	Unconverted contrast	11.6±0.5
Vehicle Control	11.5±0.5	216-2	5.5(52％)
216-3	8.8(23％)	216-2	5.5(52％)
216-3	8.8(23％)	216-4	7.4(36％)
216-5	5.8(50％)	216-4	7.4(36％)
216-5	5.8(50％)	216-8	8.4(27％)
216-10	5.1(56％)	216-8	8.4(27％)
216-10	5.1(56％)	216-11	10.1(19％)
216-12	9.3(19％)	216-11	10.1(19％)
216-12	9.3(19％)	216-13	6.4(44％)
216-15	8.8(23％)	216-13	6.4(44％)
216-15	8.8(23％)	216-16	9.7(16％)
216-17	6.4(44％)	216-16	9.7(16％)
216-17	6.4(44％)	216-19	8.7(24％)
216-21	3.2(72％)	216-19	8.7(24％)
216-21	3.2(72％)	216-24	9.4(18％)
216-26	9.3(19％)	216-24	9.4(18％)
216-26	9.3(19％)	216-29	7.1(38％)
216-30	8.2(29％)	216-29	7.1(38％)
216-30	8.2(29％)	216-32	9.3(19％)
216-34	7.1(38％)	216-32	9.3(19％)
216-34	7.1(38％)	216-35	7.8(32％)
216-38	7.1(38％)	216-35	7.8(32％)
216-38	7.1(38％)	216-42	8.1(30％)
216-44	9.4(18％)	216-42	8.1(30％)
216-44	9.4(18％)	216-45	10.2(11％)

Table 10. is expressed the PPO activity of modifying PPO gene potato system

Strain	The OD-410/ gram
	The OD-410/ gram		Microtubers (% of minimizing)	Tubercle (% of minimizing)
	Unconverted contrast	24.59±2.22	Microtubers (% of minimizing)	Tubercle (% of minimizing)	20.07±1.21
Vehicle Control	Unconverted contrast	24.59±2.22	24.59±3.36	19.55±1.43	20.07±1.21
Vehicle Control	314-1	2.36(90％)	24.59±3.36	19.55±1.43	17.8(11％)
314-2	314-1	2.36(90％)	41.52(-76％)	21.3(-7％)	17.8(11％)
314-2	314-4	18.40(22％)	41.52(-76％)	21.3(-7％)	5.4(73％)
314-5	314-4	18.40(22％)	8.49(64％)	19.1(4％)	5.4(73％)
314-5	314-7	16.04(32％)	8.49(64％)	19.1(4％)	16(20％)
314-8	314-7	16.04(32％)	14.86(37％)	17(15％)	16(20％)
314-8	314-9	5.43(77％)	14.86(37％)	17(15％)	4.3(78％)
314-12	314-9	5.43(77％)	19.35(18％)	19.6(2％)	4.3(78％)
314-12	314-13	18.17(23％)	19.35(18％)	19.6(2％)	15.4(23％)
314-14	314-13	18.17(23％)	18.64(21％)	17.32(13％)	15.4(23％)
314-14	314-16	13.92(41％)	18.64(21％)	17.32(13％)	18.2(9％)
314-17	314-16	13.92(41％)	5.19(78％)	2.4(88％)	18.2(9％)
314-17	314-20	26.66(-13％)	5.19(78％)	2.4(88％)	13.2(34％)
214-21	314-20	26.66(-13％)	11.32(52％)	17.6(12％)	13.2(34％)
214-21	214-22	13.45(43％)	11.32(52％)	17.6(12％)	18.8(6％)
314-23	214-22	13.45(43％)	5.19(78％)	20.4(-2％)	18.8(6％)
314-23	314-24	15.10(36％)	5.19(78％)	20.4(-2％)	19.6(2％)
314-25	314-24	15.10(36％)	23.12(2％)	19(5％)	19.6(2％)
314-25	314-26	13.45(43％)	23.12(2％)	19(5％)	17.8(11％)
314-27	314-26	13.45(43％)	26.42(-12％)	19.4(3％)	17.8(11％)
314-27	314-28	31.85(-35％)	26.42(-12％)	19.4(3％)	19.4(3％)
314-29	314-28	31.85(-35％)	3.77(84％)	14.8(26％)	19.4(3％)
314-29	314-31	23.83(-1％)	3.77(84％)	14.8(26％)	21.2(-6％)
314-32	314-31	23.83(-1％)	28.78(-22％)	20(0％)	21.2(-6％)

The PPO activity of table 11. in the potato tubercle of the tailer sequence of the modification of expression and PPO gene-correlation

Strain	OI-410/ restrains (% of minimizing)
Strain	OI-410/ restrains (% of minimizing)	Unconverted contrast	20.6±1.3
Vehicle Control	17.9±2.1	Unconverted contrast	20.6±1.3
Vehicle Control	17.9±2.1	217-1	12.5(39.4％)
217-4	12.6(38.6％)	217-1	12.5(39.4％)
217-4	12.6(38.6％)	217-5	11.3(45.0％)
217-6	6.1(70.4％)	217-5	11.3(45.0％)
217-6	6.1(70.4％)	217-7	5.7(72.5％)
217-9	10.4(49.6％)	217-7	5.7(72.5％)
217-9	10.4(49.6％)	217-10	15.2(26.3％)
217-11	15.2(26.3％)	217-10	15.2(26.3％)
217-11	15.2(26.3％)	217-12	6.6(67.9％)
217-14	15.4(25.4％)	217-12	6.6(67.9％)
217-14	15.4(25.4％)	217-15	13.5(34.6％)
217-16	6.0(71％)	217-15	13.5(34.6％)
217-16	6.0(71％)	217-17	9.7(53.0％)
217-19	8.6(58.4％)	217-17	9.7(53.0％)
217-19	8.6(58.4％)	217-21	14.2(31.1％)
217-22	9.7(53.0％)	217-21	14.2(31.1％)
217-22	9.7(53.0％)	217-23	15.2(26.3％)
217-24	8.2(60.1％)	217-23	15.2(26.3％)
217-24	8.2(60.1％)	217-25	11.9(42.2％)
217-26	3.1(84.8％)	217-25	11.9(42.2％)
217-26	3.1(84.8％)	217-27	6.2(69.9％)
217-29	7.2(65.1％)	217-27	6.2(69.9％)

The unmarked conversion that table 12. carries out with LifeSupport carrier+pSIM011

Plant	Cotransformation	Only underlined	Only interesting gene	Unconverted
Plant	Cotransformation	Only underlined	Only interesting gene	Unconverted	Potato	0％	33％	11％	56％
Tobacco	20％	26％	18％	36％	Potato	0％	33％	11％	56％

Cotransformation: all be positive for GUS and NPT PCR

Only interesting gene: be positive for GUS PCR

Unconverted: the plant of all being negative for GUS and NPT PCR

Table 13. transforms potato in proper order with LifeSupport carrier and pSIM011

Time window	Cotransformation	Only underlined	Only interesting gene	Unconverted
Time window	Cotransformation	Only underlined	Only interesting gene	Unconverted	0 hour	9％	36％	9％	46％
4 hours	20％	30％	20％	30％	0 hour	9％	36％	9％	46％

Unconverted: the plant of being negative for mark and interest genes PCR

The unmarked conversion that table 14. carries out with P-DNA carrier pSIM340+LifeSupport

Plant	Cotransformation	Only underlined	Only interesting gene	Unconverted
Plant	Cotransformation	Only underlined	Only interesting gene	Unconverted	Potato	17％	52.8％	1.2％	29％

Cotransformation: all be positive for the PPO gene of pSIM340 and the NPT gene PCR of LifeSupport

Unconverted: the plant of all being negative for PPO and NPTII PCR

The unmarked Transformation of potato that the LifeSupport carrier that table 15. improves with pSIM340+ carries out

The LifeSupport carrier	Cotransformation	Only underlined	Only interesting gene	Unconverted
The LifeSupport carrier	Cotransformation	Only underlined	Only interesting gene	Unconverted	pSIM346	0％	0％	4％	96％
pSIM350	10％	10％	29％	51％	pSIM346	0％	0％	4％	96％
pSIM350	10％	10％	29％	51％	pSIM401Ω	6％	34％	5％	55％
pSIM341Ω	16％	23％	7％	54％	pSIM401Ω	6％	34％	5％	55％

Unconverted: the plant of all being negative for PPO and NPTII PCR

The unmarked Transformation of potato that table 16. carries out with the single edaphic bacillus bacterial strain that contains pSIM356 and pSIM363

Plant	Cotransformation	Only underlined	Only interesting gene	Unconverted
Plant	Cotransformation	Only underlined	Only interesting gene	Unconverted	Tobacco	50％	15％	19％	16％
Potato	22％	5％	15％	58％	Tobacco	50％	15％	19％	16％

Cotransformation: all be positive for the gus gene of pSIM356 and the NPT gene PCR of LifeSupport

Unconverted: the plant of all being negative for PPO and NPTII PCR

SEQ ID Nos

SEQ ID No.:1: potato P-DNA.The runic underscore part of SEQ ID NO.1 is represented a left side (5 '-) and right (3 '-) edge sample sequence of P-DNA respectively.

SEQ ID No.:2: the P-DNA of wheat

SEQ ID No.:3:IPT expression of gene box

SEQ ID No.:4: two carrier pSIM111

SEQ ID No.:5: the gene that the saccharase of potato suppresses

SEQ ID No.:6: the GBSS promotor of potato

SEQ ID No.:7: the terminator of the ubiquitin of potato-3 gene

SEQ ID No.:8: with the potato leader sequence of R1 gene-correlation

SEQ ID No.:9: the ubiquitin intron of potato

SEQ ID No.:10: with the justice of the leader sequence of R1 gene-correlation and the expression cassette of antisense copy

SEQ ID No.:11: transcribed spacer

SEQ ID No.:12: with the justice of the leader sequence of R1 gene-correlation and the selectable expression cassette of antisense copy

SEQ ID No.:13: long potato GBSS promotor

SEQ ID No.:14: with the justice of the leader sequence of R1 gene-correlation and the selectable expression cassette of antisense copy

SEQ ID No.:15: with the justice of the leader sequence of R1 gene-correlation and the selectable expression cassette of antisense copy

SEQ ID No.:16: with the potato tailer sequence of R1 gene-correlation

SEQ ID No.:17: with the justice of the tailer sequence of R1 gene-correlation and the expression cassette of antisense copy

SEQ ID No.:18: with the justice of the tailer sequence of R1 gene-correlation and the expression cassette of antisense copy

SEQ ID No.:19: with the justice of the tailer sequence of R1 gene-correlation and the expression cassette of antisense copy

SEQ ID No.:20: with the justice of the tailer sequence of R1 gene-correlation and the expression cassette of antisense copy

SEQ ID No.:21: with the potato leader sequence of L glucosan phosphorylase gene-correlation

SEQ ID No.:22: with the potato tailer sequence of L glucosan phosphorylase gene-correlation

SEQ ID No.:23: with the justice of the leader sequence of L glucosan phosphorylase gene-correlation and the expression cassette of antisense copy

SEQ ID No.:24: with the justice of the leader sequence of L glucosan phosphorylase gene-correlation and the selectable expression cassette of antisense copy

SEQ ID No.:25: with the justice of the leader sequence of L glucosan phosphorylase gene-correlation and the selectable expression cassette of antisense copy

SEQ ID No.:26: with the justice of the leader sequence of L glucosan phosphorylase gene-correlation and the selectable expression cassette of antisense copy

SEQ ID No.:27: the PPO gene of potato

SEQ ID No.:28: the potato PPO gene of the non-activity of modification

SEQ ID No.:29: with the potato tailer sequence of PPO gene-correlation

SEQ ID No.:30: with the justice of the tailer sequence of PPO gene-correlation and the expression cassette of antisense copy

SEQ ID No.:31: with the justice of the tailer sequence of PPO gene-correlation and the selectable expression cassette of antisense copy

SEQ ID No.:32: with the justice of the tailer sequence of PPO gene-correlation and the selectable expression cassette of antisense copy

SEQ ID No.:33: with the justice of the tailer sequence of PPO gene-correlation and the selectable expression cassette of antisense copy

SEQ ID No.:34: the potato tailer sequence relevant with starch branch enzyme gene

SEQ ID No.:35: the potato tailer sequence relevant with starch branch enzyme gene

The virD2 expression of gene box of SEQ ID No.:36: Ω sudden change

SEQ ID No.:37: the salt tolerance gene Pst1 of potato

SEQ ID No.:38: the salt tolerance gene Pst2 of potato

SEQ ID No.:39: the salt tolerance gene Pst3 of potato

SEQ ID No.:40: the specific promoter of potato tuber

SEQ ID No.:56: the terminator of yeast ADH

SEQ ID No.:94: the left hand edge sample sequence of wheat

SEQ ID No.:95: the right hand edge sample sequence of wheat

SECID1

GTTTACAGTACCATATATCCTGTCAGAGGTATAGAGGCATGACTGGCATGATCACTAAATTGATGCCCACAGAGGAGACTTATAAC

CTACAGGGGCACGTAGTTCTAGGACTTGAAAGTGACTGACCGTAGTCCAACTCGGTATAAAGCCTACTCCCAACTAAATATATGAA

ATTTATAGCATAACTGCAGATGAGCTCGATTCTAGAGTAGGTACCGAGCTCGAATTCCTTACTCCTCCACAAAGCCGTAACTGAAG

CGACTTCTATTTTTCTCAACCTTCGGACCTGACGATCAAGAATCTCAATAGGTAGTTCTTCATAAGTGAGACTATCCTTCATAGCT

ACACTTTCTAAAGGTACGATAGATTTTGGATCAACCACACACACTTCG TTTACACCGGTATATATCCTGCCA

SEQID2

TGGCAGGATATATGAGTGTGTAAACAACCATAATCAGGCTGTAATTATCAAGAGAACTAATGACAAGAAGCAGAGCTTATCAAGTG

TTTCGTCCAGCTGTAACATGGGCACAAAAGCTTGCTTGATGCATGTCTGGCTTTTCAAAGAGCAATGTATTCTCAGGTACCTGCAC

GTTTGATCCCCTACCACGTACAAGACGAGCAGAAAGGACATGTCTGCAGAAACTTAGACACATCCATTGCAGACTCGTTCCGAAGC

ATCAGGAGAGTAGTCAGCAATGGTCATCTGCTGATGTAAATTAATTGATTGTTGGTAATCAAATTTTAACAGCAATATATATAATA

TATCAATAGTATATTGAACTATGAAAGACTGTAATCATATATAACAGCATACAAATTGTCGTGGAAACAAGAGGAGCTCATCAAGT

GTTTAGTTCAGAAATAGCTAACCAAGAATGCAATATAAATAGGGGTACTGAGCTCCCTTCAAAATTACTAACTTCAGAATAGCTAA

CCAAGAATGCAATGGCATTGCATAATTTAAACAACTGTCAGCACCAATCTCTGACTGAAGGCAGTTTACCCATTCAGAAGAGCACA

CATTTTCTGAACGACAACTCTGAGCGGGGATTGTTGACAGCAGCAATTAATCTGGCCTCAAGATGGTTTCCAACAACATAGATCAG

ATACAGCACTCAAGCACCCAATAATCAGCCAGTACTGATCTGGTTACCACTGCAATTGATTAACAGATGAACTGTGAAATTAAGAT

TTAACTGACAGTAATATATACCAGTTGGCAGGATATATCCCTCTGTAAAC

SEQID3

CTGCAGCCAAAGCACATACTTATCGATTTAAATTTCATCGAAGAGATTAATATCGAATAATCATATACATACTTTAAATACATAAC

AAATTTTAAATACATATATCTGGTATATAATTAATTTTTTAAAGTCATGAAGTATGTATCAAATCACATATGGAAAAAAATTAACT

ATTCATAATTTAAAAAAATAGAAAAGATACATCTAGTGAAATTAGGTGCATGTATCAAATACATTAGGAAAAGGGCATATTCTTGA

TCTAGATAATTAACGATTTTGATTTATGTATAATTTCCAAATGAAGGTTTATATCTACTTCAGAAATAACAATATACTTTTATCAG

AACATTCAACAAAGTAACAACCAACTAGAGTGAAAAATACACATTGTTCTCTAAACATACAAAATTGAGAAAAGAATCTCAAAATT

TAGAGAAACAAATCTGAATTTCTAGAAGAAAAAAATAATTATGCACTTTGCTATTGCTCGAAAAATAAATGAAAGAAATTAGACTT

TTTTAAAAGATGTTAGACTAGATATACTCAAAAGCTATCAAAGGAGTAATATTCTTCTTACATTAAGTATTTTAGTTACAGTCCTG

TAATTAAAGACACATTTTAGATTGTATCTAAACTTAAATGTATCTAGAATACATATATTTGAATGCATCATATACATGTATCCGAC

ACACCAATTCTCATAAAAAGCGTAATATCCTAAACTAATTTATCCTTCNAGTCAACTTAAGCCCAATATACATTTTCATCTCTAAA

GGCCCAAGTGGCACAAAATGTCAGGCCCAATTACGAAGAAAAGGGCTTGTAAAACCCTAATAAAGTGGCACTGGCAGAGCTTACAC

TCTCATTCCATCAAACAAAGAAACCCTAAAGCCGCAGCGCCACTGATTTCTCTCCTCCAGGCGAAGATGCAGATCTTCGTGAAGAC

CCTAACGGGGAAGACGATCACCCTAGAGGTTGAGTCTTCCGACACCATCGACAATGTCAAAGCCAAGATCCAGGACAAGGAAGGGA

TTCCCCCAGACCAGCAGCGTTTGATTTTCGCCGGAAAGCAGCTTGAGGATGGTCGTACTCTTGCCGACTACAACATCCAGAAGGAG

TCAACTCTCCATCTCGTGCTCCGTCTCCGTGGTGGTGGATCCATGGACCTGCATCTAATTTTCGGTCCAACTTGCACAGGAAAGAC

GACGACCGCGATAGCTCTTGCCCAGCAGACAGGGCTTCCAGTCCTTTCGCTTGATCGGGTCCAATGCTGTCCTCAACTATCAACCG

GAAGCGGACGACCAACAGTGGAAGAACTGAAAGGAACGACGCGTCTCTACCTTGATGATCGGCCTCTGGTGGAGGGTATCATCGCA

GCCAGCAAGCTCATCATAGGCTGATCGAGGAGGTGTATAATCATGAAGGCCAACGGCGGGCTTATTCTTGAGGGAGGATCCACCTC

GTTGCTCAACTGCATGGCGCGAAACAGCTATTGGAGTGCAGATTTTCGTTGGCATATTATTCGCCACAAGTTACCCGACCAAGAGA

CCTTCATGAAAGCGGCCAAGGCCAGAGTTAAGCAGATGTTGCACCCCGCTGCAGGCCATTCTATTATTCAAGAGTTGGTTTATCTT

TGGAATGAACCTCGGCTGAGGCCCATTCTGAAAGAGATCGATGGATATCGATATGCCATGTTGTTTGCTAGCCAGAACCAGATCAC

GGCAGATATGCTATTGCAGCTTGACGCAAATATGGAAGGTAAGTTGATTAATGGGATCGCTCAGGAGTATTTCATCCATGCGCGCC

AACAGGAACAGAAATTCCCCCAAGTTAACGCAGCCGCTTTCGACGGATTCGAAGGTCATCCGTTCGGAATGTATTAGGTTACGCCA

GCCCTGCGTCGCACCTGTCTTCATCTGGATAAGATGTTCGTAATTGTTTTTGGCTTTGTCCTGTTGTGGCAGGGCGGCAAATACTT

CCGACAATCCATCGTGTCTTCAAACTTTATGCTGGTGAACAAGTCTTAGTTTCCACGAAAGTATTATGTTAAATTTTAAAATTTCG

ATGTATAATGTGGCTATAATTGTAAAAATAAACTATCGTAAGTGTGCGTGTTATGTATAATTTGTCTAAATGTTTAATATATATCA

TAGAACGCAATAAATATTAAATATAGCGCTTTTATGAAATATAAATACATCATTACAAGTTGTTTATATTTCGGGTGGACTAGTTT

TTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTT

GATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAAT

CTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTA

TCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTA

AAAAAATAGGAATTC

SEQID4

AGCTTTGGCAGGATATATACCGGTGTAAACGAAGTGTGTGTGGTTGATCCAAAATCTATCGTACCTTTAGAAAGTGTAGCTATGAA

GGATAGTCTCACTTATGAAGAACTACCTATTGAGATTCTTGATCGTCAGGTCCGAAGGTTGAGAAAAATAGAAGTCGCTTCAGTTA

CGGCTTTGTGGAGGAGTAAGGGTACCTACTCTAGAATCGAGCTCATCGTTATGCTATAAATTTCATATATTTAGTTGGGAGTAGGC

TTTATACCGAGTTGGACTACGGTCAGTCACTTTCAAGTCCTAGAACTACGTGCCCCTGTAGGTTATAAGTCTCCTCTGTGGGCATC

AATTTAGTGATCATGCCAGTCATGCCTCTATACCTCTGACAGGATATATGGTACTGTAAACACTAGTTGTGAATAAGTCGCTGTGT

ATGTTTGTTTGAGATCTCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTC

CATTTGTATGTGGTCACCTATCTCGAGCATGCCAACCACAGGGTTCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCCGCTGC

TATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGC

CGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACG

CCATGAACAAGAGCGCCGCCGCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAA

CTGCACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGA

CCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCCGAGC

GCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCCGGCCGCATGGTGTTGACC

GTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGT

GAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAG

AGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGG

CGGCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAGGAACAAGCATG

AAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTCGA

GCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCT

TGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTAT

ATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAG

ACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGC

CCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCA

TCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTG

CTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGA

TGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGT

ACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCA

GAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAG

AAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGA

CACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGA

CCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGC

TAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAG

GCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATC

CGGCCCGGTACAAATCGGCGCGCCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCAACGCATCG

AGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGCAGCCGGTCCG

CCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCG

CAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGC

ACGTAGAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAA

TCCATGAACCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCTGCCG

GCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACGA

AGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGG

CGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCA

CCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGAT

GGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCA

AATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGG

CGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTCTCTTTC

CTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGATACGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGG

AACCGGTCACACATGTAAGTGACTGATATAAAAGAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCT

TAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTCCGCT

CCCTACGCCCCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGG

CGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAA

ACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCA

GCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCA

GAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACACATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTC

CGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTAT

CCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTG

GCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATA

AAGATACCAGAGGTTTCCCCCTGGAAGCTCCCTCGCGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTC

TCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT

GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATC

GCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT

ACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGCAAAAAGAGTTGCTAGCTCTTGATCC

GGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCC

TTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATTCTAGGTACTAAAACA

ATTCATCCAGTAAAATATAATATTTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCT

TCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCAAGATCAATAAA

GCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCG

TCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTA

TTCAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGAGTGAAAGAGCCT

GATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGACGCCATCGGCCTCAC

TCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGCATCATG

TCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCTGTCCGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAG

CTTATATACCTTAGCAGGAGACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGGTGATATTC

TCATTTTAGCCATTTATTATTTCCTTCCTCTTTTCTACAGTATTTAAAGATACCCCAAGAAGCTAATTATAACAAGACGAACTCCA

ATTCACTGTTCCTTGCATTCTAAAACCTTAATAACCAGAAAACAGCTTTTTCAAAGTTGTTTTCAAAGTTGGCGTATAACATAGTA

TCGACGGAGCCGATTTTGAAACCGCGGATCCTGCAGCCAAAGCACATACTTATCGATTTAAATTTCATCGAAGAGATTAATATCGA

ATAATCATATACATACTTTAAATACATAACAAATTTTAAATACATATATCTGGTATATAATTAATTTTTTAAAGTCATGAAGTATG

TATCAAATACACATATGGAAAAAATTAACTATTCATAATTTAAAAAATAGAAAAGATACATCTAGTGAAATTAGGTGCATGTATCA

AATACATTAGGAAAAGGGCATATATCTTGATCTAGATAATTAACGATTTTGATTTATGTATAATTTCCAAATGAAGGTTTATATCT

ACTTCAGAAATAACAATATACTTTTATCAGAACATTCAACAAAGTAACAACCAACTAGAGTGAAAAATACACATTGTTCTCTAAAC

ATACAAAATTGAGAAAAGAATCTCAAAATTTAGAGAAACAAATCTGAATTTCTAGAAGAAAAAAATAATTATGCACTTTGCTATTG

CTCGAAAAATAAATGAAAGAAATTAGACTTTTTTAAAAGATGTTAGACTAGATATACTCAAAAGCTATCAAAGGAGTAATATTCTT

CTTACATTAAGTATTTTAGTTACAGTCCTGTAATTAAAGACACATTTTAGATTGTATCTAAACTTAAATGTATCTAGAATACATAT

ATTTGAATGCATCATATACATGTATCCGACACACCAATTCTCATAAAAAGCGTAATATCCTAAACTAATTTATCCTTCAAGTCAAC

TTAAGCCCAATATACATTTTCATCTCTAAAGGCCCAAGTGGCACAAAATGTCAGGCCCAATTACGAAGAAAAGGGCTTGTAAAACC

CTAATAAAGTGGCACTGGCAGAGCTTACACTCTCATTCCATCAACAAAGAAACCCTAAAAGCCGCAGCGCCACTGATTTCTCTCCT

CCAGGCGAAGATGCAGATCTTCGTGAAGACCCTAACGGGGAAGACGATCACCCTAGAGGTTGAGTCTTCCGACACCATCGACAATC

TCAAAGCCAAGATCCAGGACAAGGAAGGGATTCCCCCAGACCAGCAGCGTTTGATTTTCGCCGGAAAGCAGCTTGAGGATGGTCGT

ACTCTTGCCGACTACAACATCCAGAAGGAGTCAACTCTCCATCTCGTGCTCCGTCTCCGTGGTGGTGGATCCATGGACCTGCATCT

AATTTTCGGTCCAACTTGCACAGGAAAGACGACGACCGCGATAGCTCTTGCCCAGCAGACAGGGCTTCCAGTCCTTTCGCTTGATC

GGGTCCAATGCTGTCCTCAACTATCAACCGGAAGCGGACGACCAACAGTGGAAGAACTGAAAGGAACGACGCGTCTCTACCTTGAT

GATCGGCCTCTGGTGGAGGGTATCATCGCAGCCAAGCAAGCTCATCATAGGCTGATCGAGGAGGTGTATAATCATGAGGCCAACGG

CGGGCTTATTCTTGAGGGAGGATCCACCTCGTTGCTCAACTGCATGGCGCGAAACAGCTATTGGAGTGCAGATTTTCGTTGGCATA

TTATTCGCCACAAGTTACCCGACCAAGAGACCTTCATGAAAGCGGCCAAGGCCAGAGTTAAGCAGATGTTGCACCCCGCTGCAGGC

CATTCTATTATTCAAGAGTTGGTTTATCTTTGGAATGAACCTCGGCTGAGGCCCATTCTGAAAGAGATCGATGGATATCGATATGC

CATGTTGTTTGCTAGCCAGAACCAGATCACGGCAGATATGCTATTGCAGCTTGACGCAAATATGGAAGGTAAGTTGATTAATGGGA

TCGCTCAGGAGTATTTCATCCATGCGCGCCAACAGGAACAGAAATTCCCCCAAGTTAACGCAGCCGCTTTCGACGGATTCGAAGGT

CATCCGTTCGGAATGTATTAGGTTACGCCAGCCCTGCGTCGCACCTGTCTTCATCTGGATAAGATGTTCGTAATTGTTTTTGGCTT

TGTCCTGTTGTGGCAGGGCGGCAAATACTTCCGACAATCCATCGTGTCTTCAAACTTTATGCTGGTGAACAAGTCTTAGTTTCCAC

GAAAGTATTATGTTAAATTTTAAAAATTTCGATGTATAATGTGGCTATAATTGTAAAAATAACTATCGTAAGTGTGCGTGTTATGT

ATAATTTGTCTAAATGTTTAATATATATCATAGAACGCAATAAATATTAAATATAGCGCTTTTATGAAATATAAATACATCATTAC

AAGTTGTTTATATTTCGGGTGGACTAGTTTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGA

GTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGC

TTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAA

TTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTT

TATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGGAATTCTATCCGCGGTGATCACAGGCAGCAACGCTCTGTCATCGTTA

CAATCAACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACAT

GAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGC

CGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGA

SEQID5

ATGAGAAATTTATTCCCCATATTGATGCTAATCACCAATTTGGCACTCAACAACGATAACAACAACAACAACAACAACAACAATAA

TTATAATCTCATACACGCAACGTGTAGGGAGACCCCATATTACTCCCTATGTCTCACCACCCTACAATCCGGTCCACGTAGTAACG

AGGTTGAGGGTGGTGATGCCATCACCACCCTAGGCCTCATCATGGTGGACGCGGTGAAATCAAAGTCCATAGAAATAATGGAAAAA

ATAAAAGAGCTAGAGAAATCGAACCCTGAGTGGCGGGCCCCACTTAGCCAGTGTTACGTGGCGTATAATGCCGTCCTACGAGCCGA

TGTAACGGTAGCCGTTGAAGCCTTAAAGAAGGGTGCCCCCAAATTTGCTGAAGATGGTATGGATGATGTTGTTGCTGAAGCACAAA

CTTGTGAGTATAGTTTTAATTATTATAATAAATTGGATTTTCCAATTTCTAATTTGAGTAGGGAAATAATTGAACTATCAAAAGTT

GCTAAATCCATAATTAGAATGTTATTATGA

SEQID6

GAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTT

ATTACTATAATAATATTTTAATTAACACGACACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATA

CTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGT

CGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTG

AAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAACAAGTGGTAACTTTTACTCATCT

CCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCA

TCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTC

TTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGGTAG

SEQID7

TTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTT

TTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCA

ATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAAC

TATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAG

TAAAAAAATAG

SEQID8

ACCTTATTTCACTACCACTTTCCACTCTCCAATCCCCATACTCTCTGCTCCAATCTTCATTTTGCTTCGTGAATTCATCTTCATCG

AATTTCTCGACGCTTCTTCGCTAATTTCCTCGTTACTTCACTAAAAATCGACGTTTCTAGCTGAACTTGAGTGAATTAAGCCAGTG

GGAGGAT

SEQID9

GTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAG

TTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTT

TCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTAC

ATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTT

TGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAA

AAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCC

CTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAG

SEQID10

GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTA

TCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTT

TAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAA

TACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTG

TCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCACCTTATT

TCACTACCACTTTCCACTCTCCAATCCCCATACTCTCTGCTCCAATCTTCATTTTGCTTCGTGAATTCATCTTCATCGAATTTCTC

GACGCTTCTTCGCTAATTTCCTCGTTACTTCACTAGAAATCGACGTTTCTAGCTGAACTTGAGTGAATTAAGCCAGTGGGAGGATG

AATTCAAGGTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCCTTA

ATTTTGAGTTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAAT

TGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGA

GTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTT

CGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTT

TTGATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGT

GAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCATCCTCCCACTGGCTT

AATTCACTCAAGTTCAGCTAGAAACGTCGATTTCTAGTGAAGTAACGAGGAAATTAGCGAAGAAGCGTCGAGAAATTCGATGAAGA

TGAATTCACGAAGCAAAATGAAGATTGGAGCAGAGAGTATGGGGATTGGAGAGTGGAAAGTGGTAGTGAAATAAGGTAAGCTTTTG

ATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTT

TTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATC

AATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAA

CTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATA

GTAAAAAAATAGTCTAGA

SEQID11

GTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAA

CGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTG

AATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAG

SEQID12

AATTCGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGG

TGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGA

TAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGATATCATCC

TCCCACTGGCTTAATTCACTCAAGTTCAGCTAGAAACGTCGATTTCTAGTGAAGTAACGAGGAAATTAGCGAAGAAGCGTCGAGAA

ATTCGATGAAGATGAATTCACGAAGCAAAATGAAGATTGGAGCAGAGAGTATGGGGATTGGAGAGTGGAAAGTGGTAGTGAAATAA

GGTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAAGGGTAATTTAGAGTTTTTTTTGCTATA

TGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCT

ATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTA

TGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTT

GAGTATAATATAGTAAAAAAATAGTCTAGA

SEQID13

ATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATA

TTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGGTAGATTCCCCTTTTTGTAGACCACACATCAC

SEQID14

TCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCTCATATTC

TAGTTGTATGTTGTTCAGAGAAGACCACAGATGTGATCATATTCTCATTGTATCAGATCTGTGACCACTTACCTGATACCTCCCAT

GAAGTTACCTGTATGATTATACGTGATCCAAAGCCATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGAGAAGTAGGAAT

TGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCTGGGTATGATATAGGGAGAAATGTGTAAACATTGTACTA

TATATAGTATATACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAAAGAAGGAATTCAAGGTTAGAAATC

TTCTCTATTTTTGGTTTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAGTTTTTTTTCG

GTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGT

TTTGATGAAAAAGCCCCTAGTTTGAGATTTTT1TTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACATTTGTTTGA

TGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTCTGAA

GGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAA

ATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTG

AGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCCTTCTTTGATGCTGCGGTATTATTCAGTGCATAA

TGCAATACATAATGCGTGTGTATATACTATATATAGTACAATGTTTACACATTTCTCCCTATATCATACCCAGCTAATTTAGCTTT

TACAAAGTTTTTCTTATTATTCCTCATATTGCAATTCCTACTTCTCACTTCTCCTCCAATAGCTGAAGGTGAACATGATGTGATGG

CTTTGGATCACGTATAATCATACAGGTAACTTCATGGGAGGTATCAGGTAAGTGGTCACAGATCTGATACAATGAGAATATGATCA

CATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATGAAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTC

TTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTT

ATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTG

TTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCA

GGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA

SEQID15

GAAGTTACCTGTATGATTATACGTGATCCAAAGCCATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGA1AAGTAGGAAT

TATATAGTATATACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAAAGAAGGAATTCGTGGTAACTTTTA

CTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCA

TATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCT

CTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGATATCCTTCTTTGATGCTGCGGTATTA

TTCAGTGCATAATGCAATACATAATGCGTGTGTATATACTATATATAAGTACATGTTTACACATTTCTCCCTATATCATACCCAGC

TAATTTAGCTTTTACAAAGTTTTTCTTATTATTCCTCATATTGCAATTCCTACTTCTCACTTCTCCTCCAATAGCTGAAGGTGAAC

ATGATGTGATGGCTTTGGATCACGTATAATCATACAGGTAACTTCATGGGAGGTATCAGGTAAGTGGTCACAGATCTGATACAATG

AGAATATGATCACATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATGAAAGCTTTTGATTTTAATGTTTAGCAAATGTCCT

ATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGG

GATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTA

GTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAA

ATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA

SEQID16

TCATATTCTAGTTGTATGTTGTTCAGAGAAGACCACAGATGTGATCATATTCTCATTGTATCAGATCTGTGACCACTTACCTGATA

CCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAAGCCATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGAGAA

GTAGGAATTGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCTGGGTATGATATAGGGAGAAATGTGTAAACA

TTGTACTATATATAGTATATACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAAAGAAG

SEQID17

TTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACATTTGTTTGA

GGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA

SEQID18

TTCAGTGCATAATGCAATACATAATGCGTGTGTATATACTATATATAGTACAATGTTTACACATTTCTCCCTATATCATACCCAGC

AGAATATCATCACATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATGAAAGCTTTTGATTTTAATGTTTAGCAAATGTCCT

SEQIDl9

TCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAACAAGTGGTAACTTTTAC

TCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCAT

ATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTC

TTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGGTAGATTCCCCTTTTTGTAGACCACACA

TCACGGATCCTCATATTCTAGTTGTATGTTGTTCAGAGAAGACCACAGATGTGATCATATTCTCATTGTATCAGATCTGTGACCAC

TTACCTGATACCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAAGCCATCACATCATGTTCACCTTCAGCTATTGGAGGA

GAAGTGAGAAGTAGGAATTGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCTGGGTATGATATAGGGAGAAA

TGTGTAAACATTGTACTATATATAGTATATACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAAAGAAGG

GAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCCTTCTTTGATGCTGCG

GTATTATTCAGTGCATAATGCAATACATAATGCGTGTGTATATACTATATATAGTACAATGTTTACACATTTCTCCCTATATCATA

CCCAGCTAATTTAGCTTTTACAAAGTTTTTCTTATTATTCCTCATATTGCAATTCCTACTTCTCACTTCTCCTCCAATAGCTGAAG

GTGAACATGATGTGATGGCTTTGGATCACGTATAATCATACAGGTAACTTCATGGGAGGTATCAGGTAAGTGGTCACAGATCTGAT

ACAATGAGAATATGATCACATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATGAAAGCTTTTGATTTTAATGTTTAGCAAA

TGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAAC

CCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCT

TTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGAT

CGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA

SEQID20

SEQID21

TTAGAGTGTGGGTAAGTAATTAAGTTAGGGATTTGTGGGAAATGGACAAATATAAGAGAGTGCAGGGGAGTAGTGCAGGAGATTTT

CGTGCTTTTATTGATAAATAAAAAAAGGGTGACATTTAATTTCCACAAGAGGACGCAACACAACACACTTAATTCCTGTGTGTGAA

TCAATAATTGACTTCTCCAATCTTCATCAATAAAATAATTCACAATCCTCACTCTCTTATCACTCTCATTCGAAAAGCTAGATTTG

CATAGAGAGCACAAA

SEQID22

GAGGGGGAAGTGAATGAAAAATAACAAAGGCACAGTAAGTAGTTTCTCTTTTTATCATGTGATGAAGGTATATAATGTATGTGTAA

GAGGATGATGTTATTACCACATAATAAGAGATGAAGAGTCTCATTTTCTGCTTAAAAAAACAATTCACTGGC

SEQID23

TCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGATGTCAAGTGGTAGCGGTAGGAAGGGAGTTGGTTCAGTTTTT

TCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCGAGTGTGG

GTAAGTAATTAAGTTAGGGATTTGTGGGAAATGGACAAATATAAGAGAGTGCAGGGGAGTAGTGCAGGAGATTTTCGTGCTTTTAT

TGATAAATAAAAAAAGGGTGACATTTAATTTCCACAAGAGGACGCAACACACAACACACTTAATTCCTGTGTGAATCAATAATTGA

CTTCTCCAATCTTCATCAATAAAATAATTCACAATCCTCACTCTCTTATCACTCTCATTCGAAAAGCTAGATTTGCATAGAGAGCA

CAGAATTCAAGGTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTTTAGATTCTCG1ATTAGCTAATCAGGTGCTGTTATAGCCC

TTAATTTTGAGTTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTAC

AATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATT

AGAGTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTT

TTTCGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCT

GTTTTGATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAG

TGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCTGTGCTCTCTATG

CAAATCTAGCTTTTCGAATGAGAGTGATAAGAGAGTGAGGATTGTGAATTATTTTATTGATGAAGATTGGAGAAGTCAATTATTGA

TTCACACACAGGAATTAAGTGTGTTGTGTTGCGTCCTCTTGTGGAAATTAAATGTCACCCTTTTTTTATTTATCAATAAAAGCACG

AAAATCTCCTGCACTACTCCCCTGCACTCTCTTATATTTGTCCATTTCCCACAAATCCCTAACTTAATTACTTACCCACACTCTAA

GCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGAT

TTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTT

GGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGAT

TATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTA

TAATATAGTAAAAAAATAGTCTAGA

SEQID24

TGATAAATAAAAAAAGGGTGACATTTAATTTCCACAAGAGGACGCAACACAACACACTTAATTCCTGTGTGTGAATCAATAATTGA

CAGAATTCGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTA

TGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGG

TGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGATATCT

GTGCTCTCTATGCAAATCTAGCTTTTCGAATGAGAGTGATAAGAGAGTGAGGATTGTGAATTATTTTATTGATGAAGATTGGAGAA

GTCAATTATTGATTCACACACAGGAATTAAGTGTGTTGTGTTGCGTCCTCTTGTGGAAATTAAATGTCACCCTTTTTTTATTTATC

AATAAAAGCACGAAAATCTCCTGCACTACTCCCCTGCACTCTCTTATATTTGTCCATTTCCCACAAATCCCTAACTTAATTACTTA

CCCACACTCTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTT

TGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTT

CTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTG

CAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCA

TTGGGTTGAGTATATATAGTAAAAAATAGTCTAGA

5EQID25

TCATTCTCCTCCATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCAT

TCACGGATCCGAGTGTGGGTAAGTAATTAAGTTAGGGATTTGTGGGAAATGGACAAATATAAGAGAGTGCAGGGGAGTAGTGCAGG

AGATTTTCGTGCTTTTATTGATAAATAAAAAAAGGGTCACATTTAATTTCCACAAGAGGACGCAACACAACACACTTAATTCCTGT

GTGTGAATCAATAATTGACTTCTCCAATCTTCATCAATAAAATAATTCACAATCCTCACTCTCTTATCACTCTCATTCGAAAAGCT

AGATTTGCATAGAGAGCACAGAATTCAAGGTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTTTAGATTCTCGAATTAGCTAAT

CAGGTGCTGTTATAGCCCTTAATTTTGAGTTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGT

TTGATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGA

TTCTAAAGGTTTAAAATTAGAGTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAA

GCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAA

ATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCG

AATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGG

ATATCTGTGCTCTCTATGCAAATCTAGCTTTTCGAATGAGAGTGATAAGAGAGTGAGGATTGTGAATTATTTTATTGATGAAGATT

GGAGAAGTCAATTATTGATTCACACACAGGAATTAAGTGTGTTGTGTTGCGTCCTCTTGTGGAAATTAAATGTCACCCTTTTTTTA

TTTATCAATAAAAGCACGAAAATCTCCTGCACTACTCCCCTGCACTCTCTTATATTTGTCCATTTCCCACAAATCCCTAACTTAAT

TACTTACCCACACTCTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGT

TTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTT

ATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATT

TGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTA

TGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA

SEQID26

AGATTTTCGTGCTTTTATTGATAAATAAAAAAAGGGTGACATTTAATTTCCACAAGAGGACGCAACACAACACACTTAATTCCTGT

AGATTTGCATAGAGAGCACAGAATTCGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATT

CTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATT

TGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTC

ATCTGTAGCTTGATATCTGTGCTCTCTATGCAAATCTAGCTTTTCGAATGAGAGTGATAAGAGAGTGAGGATTGTGAATTATTTTA

TTGATGAAGATTGGAGAAGTCAATTATTGATTCACACACAGGAATTAAGTGTGTTGTGTTGCGTCCTCTTGTGGAAATTAAATGTC

ACCCTTTTTTTATTTATCAATAAAAGCACGAAAATCTCCTGCACTACTCCCCTGCACTCTCTTATATTTGTCCATTTCCCACAAAT

CCCTAACTTAATTACTTACCCACACTCTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACG

GTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAA

GAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAAC

ACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATT

TTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA

SEQID27

ATGGCAAGCTTGTGCAATAGTAGTAGTACATCTCTCAAAACTCCTTTTACTTCTTCCTCCACTTCTTTATCTTCCACTCCTAAGCC

CTCTCAACTTTTCATCCATGGAAAACGTAACCAAATGTTCAAAGTTTCATGCAAGGTTATCAATAATAACGGTGACCAAAACGTTG

AAACGAATTCTGTTGATCGAAGAAATGTTCTTCTTGGCTTAGGTGGTCTTTATGGTGTTGCTAATGCTATACCATTAGCTGCATCC

GCTGCTCCAACTCCACCTCCTGATCTCTCGTCTTGTAGTATAGCCAGGATTAACGAAAATCAGGTGGTGCCGTACAGTTGTTGCGC

GCCTAAGCCTGATGATATGGAGAAAGTTCCGTATTACAAGTTCCCTTCTATGACTAAGCTCCGTGTCCGTCAGCCTGCTCATGAAG

CTAATGAGGAGTATATTGCCAAGTACAATCTGGCGATTAGTCGAATGAGAGATCTTGATAAGACACAACCTTTAAACCCTATTGGT

TTTAAGCAACAAGCTAATATACATTGTGCTTATTGTAATGGTGCTTATAGAATTGGTGGCAAAGAGTTACAAGTTCATAATTCTTG

GCTTTTCTTCCCGTTCCATAGATGGTACTTGTACTTCCACGAGAGAATCGTGGGAAAATTCATTGATGATCCAACTTTCGCTTTGC

CATATTGGAATTGGGACCATCCAAAGGGTATGCGTTTTCCTGCCATGTATGATCGTGAAGGGACTTCCCTTTTCGATGTAACACGT

GACCAAAGTCACCGAAATGGAGCAGTAATCGATCTTGGTTTTTTCGGCAATGAAGTCGAAACAACTCAACTCCAGTTGATGAGCAA

TAATTTAACACTAATGTACCGTCAAATGGTAACTAATGCTCCATGTCCTCGGATGTTCTTTGGTGGGCCTTATGATCTCGGGATTA

ACACTGAACTCCCGGGAACTATAGAAAACATTCCTCACGGTCCTGTCCACATCTGGTCTGGTACAGTGAGAGGTTCAACTTTGCCC

AATGGTGCAATATCAAACGGTGAGAATATGGGTCATTTTTACTCAGCTGCTTTGGACCCGGTTTTCTTTTGCCATCACAGCAATGT

GGATCGGATGTGGAGCGAATGGAAAGCGACAGGAGGGAAAAGAACAGATATCACACATAAAGGTTGGTTGAACTCCGAGTTCTTTT

TCTATGATGAAAATGAAAACCCTTACCGTGTGAAAGTCCGAGACTGTTTGGACACGAAGAAGATGGGGTATGATTATGCACCAATG

GCCACCCCGTGGCGTAACTTCAAGCCAATAACAAAAACTACAGCTGGGAAAGTGAATACAGCTTCTCTTCCGCCAGCTAGCAATGT

ATTCCCAGTGGCTAAACTCGACAAAGCAATTTCGTTTTCCATCAATAGGCCGACTTCGTCAAGGACTCAACAAGAGAAAAATGCAC

AAGAGGAGATGTTGACATTCAGTAGCATAAGATATGATAACAGAGGGTACATAAGGTTCGATGTGTTCCTGAACGTGGACAATAAT

GTGAATGCGAATGAGCTTGACAAGGCGGAGTTTGCGGGGAGTTATACTAGTTTGCCACATGTTCATAGAGCTGGTGAGACTAATCA

TATCGCGACTGTTGATTTCCAGCTGGCGATAACGGAACTGTTGGAGGATATTGGTTTGGAAGATGAAGATACTATTGCGGTGACTC

TGGTGCCAAAGAGAGGTGGTGAAGGTATCTCCATTGAAAGTGCGACGATCAGTCTTGCAGATTGTTAA

SEQTD28

TTTAAGCAACAAGCTAATATACAGTGGGCTTATGGTAATGGTGCTTATAGAATTGGTGGCAAAGAGTTACAAGTTCATAATTCTTG

ACACTGAACTCCCGGGAACTATAGGAAACATTCCTCTCGGTCCTGTCCACATCTGGTCTGGTACAGTGAGAGGTTCAACTTTGCCC

TGGTGCCAAAGAGAGGTGGTGAAGGTATCTCCATTGAAAGTGCGACGATCAGTCTTGCAGATTGTTAA

SEQID29

TTAGTCTCTATTGAATCTGCTGAGATTACACTTTGATGGATGATGCTCTGTTTTTGTTTTCTTGTTCTGTTTTTTCCTCTGTTGAA

ATCAGCTTTGTTGCTTGATTTCATTGAAGTTGTTATTCAAGAATAAATCAGTTACAATTATGTTTGGG

SEQID30

TAAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAA

TCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCTTAGTCTC

TATTGAATCTGCTGAGATTACACTTTGATGGATGATGCTCTGTTTTTGTTTTCTTGTTCTGTTTTTTCCTCTGTTGAAATCAGCTT

TGTTGCTTGATTTCATTGAAGTTGTTATTCAAGAATAAATCAGTTACAATTATGGAATTCAAGGTTAGAAATCTTCTCTATTTTTG

GTTTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAGTTTTTTTTCGGTTGTTTTGATGG

AAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAG

CCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACATTTGTTTGATGAAAAAGGCCTT

AAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTCTGAAGGCCTAAAATTTG

AGTTTCTCCGGCTGTTTTGATGAAAAAAGCCCTAAATTTGGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTTTT

CCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTT

GTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCCTTCTTTGATGCTGATCCATAATTGTAACTGATTTATTCTTGAATAA

CAACTTCAATGAAAATCAAGCAACAAAGCTGATTTCAACGAGGAAAAAACAGAACAAGAAAACAAAAACAGAGCATCATCCATCAA

AGTGTAATCTCAGCAGATTCAATAGAGACTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAA

CGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACT

AAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCA

ACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGA

TTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA

SEQID31

TGTTGCTTGATTTCATTGAAGTTGTTATTCAAGAATAAATCAGTTACAATTATGGAATTCGTGGTAACTTTTACTCATCTCCTCCA

ATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCATCTCAT

CTATTCTGATTTTGMTTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTC

TTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGATATCCTTCTTTGATGCTGATCCATAATTGTAACTGATTT

ATTCTTGAATAACAACTTCAATGAAATCAAGCAACAAAGCTGATTTCAACAGAGGAAAAAACAGAACAAGAAAACAAAAACAGAGC

ATCATCCATCAAAGTGTAATCTCAGCAGATTCAATAGAGACTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCT

CTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATT

TATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGT

GTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTC

AGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGGATATAATATAGTAAAAAAATAGTCTAGA

SEQID32

GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGATGGACCAGTACCAGTACATTAGATATTA

TCACGGATCCTTAGTCTCTATTGAATCTGCTGAGATTACACTTTGATGGATGATGCTCTGTTTTTGTTTTCTTGTTCTGTTTTTTC

CTCTGTTGAAATCAGCTTTGTTGCTTGATTTCATTGAAGTTGTTATTCAAGAATAAATCAGTTACAATTATGGAATTCAAGGTTAG

AAATCTTCTCTATTTTTGGTTTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAGTTTTT

TTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCCCC

GTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACATTTG

TTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATT

CTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGC

CCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAA

ATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCCTTCTTTGATGCTGATCCATAATTGTAAC

TGATTTATTCTTGAATAACAACTTCAATGAAATCAAGCAACAAAGCTGATTTCAACAGAGGAAAAAACAGAACAAGAAAACAAAAA

CAGAGCATCATCCATCAAAGTGTAATCTCAGCAGATTCAATAGAGACTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAG

TTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTG

TTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCT

CTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATG

CTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA

SEQID33

TTCTTCTTCTTCTTCTCAGAAAATCAATTTCTGTTTTGTTTTGTTCATCTGTAGCTTGGTAGATTCCCCTTTTTGTAGACCACACA

CTCTGTTGAAATCAGCTTTGTTGCTTGATTTCATTGAAGTTGTTATTCAAGAATAAATCAGTTACAATTATGGAATTCGTGGTAAC

TTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTG

TTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGC

TTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGATATCCTTCTTTGATGCTGATC

CATAATTGTAACTGATTTATTCTTGAATAACAACTTCAATGAAATCAAGCAACAAAGCTGATTTCAACAGAGGAAAAAACAGAACA

AGAAAACAAAAACAGAGCATCATCCATCAAAGTGTAATCTCAGCAGATTCAATAGAGACTAAGCTTTTGATTTTAATGTTTAGCAA

ATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAA

CCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATC

TTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGA

TCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAG

A

SEQID34

GTCCATGATGTCTTCAGGGTGGTAGCATTGACTGATGGCATCATAGTTTTTTTTTTAAAAGTATTTCCTCTATGCATATTATTAGT

ATCCAATAAATTTACTGGTTGTTGTACATAGAAAAAGTGCATTTGCATGTATGTGTTTCTCTGAAATTTTCCCCAGTTTTTGGTGC

TTTGCCTTTGGAGCCAAGTCTCTATATGTATAAGAAAACTAAGAACAATCACATATATCAAATATTAG

SEQID35

ACGAACTTGTGATCGCGTTGAAAGATTTGAACGCTACATAGAGCTTCTTGACGTATCTGGCAATATTGCATCAGTCTTGGCGGAAT

TTCATGTGACAACAAGGTTTGCAATTCTTTCCACTATTAGTAGTGCAACGATATACGCAGAGATGAAGTGCTGAACAAACATATGT

AAAATCGATGAATTTATGTCGAATGCTGGGACGGGCTTCAGCAGGTTTTGCTTAGT

SEQID36

CCGCGGTTTTCTCTCCATCGCGTCAGAGGCCGGTTTTCGTCGGCATCGAAGAGGGCCACTCGTTTACCGTCATTTGCCAAAGCAGC

GCAAAGGCCCATGAGTGCGGTGGTTTTGCCAGCACCCCCTTTGAAAGAGCAAAACGTCAAAAGTTGCATATTCTGATCCCGCCTGT

CCTGTGAAACGGAGTGCATTTGTATTTTTGTTCGTATAAATGTTTTTGTGATTATCGATGAGTAAAAGCGTTGTTACACTATTTTT

TATTTCAAATTCGTTATAATTAAATTGCAATTGTAGCAATTATATTCGGTTTTTCCTGTAAATATACTGTTGATTTCATATCGAGT

AGGGCTAGACTTTAATCTGTCTACCCGGGCACATTTCGTGCTGGAGTATTCAGACCTTCCGCTTTTTTTGGAGGAAGCTATGTCAA

AACACACCAGAGTCACGTCGAGTGAGACTGCCATCAACCAGCATCGATCCCTGAACGTTGAAGGGTTTAAGGTCGTGAGTGCCCGT

CTGCGATCGGCCGAGTATGAAACCTTTTCCTATCAAGCGCGCCTGCTGGGACTTTCGGATAGTATGGCAATTCGCGTTGCGGTGCG

TCGCATCGGGGGCTTTCTCGAAATAGATGCACACACCCGAGAAAAGATGGAAGCCATACTTCAGTCCATCGGAATACTCTCAAGTA

ATGTATCCATGCTTCTATCTGCCTACGCCGAAGACCCTCGATCGGATCTGGAGGCTGTGCGAGATGAACGTATTGCTTTTGGTGAG

GCTTTCGCCGCCCTCGATGGCCTACTCCGCTCCATTTTGTCCGTATCCCGGCGACGGATCGACGGTTGCTCGCTATTGAAAGGTGC

CTTGTAGCACTTGACCACGCACCTGACGGGAGAAAATTGGATGCCCGATCGCGCTCAAGTAATCATTCGCATTGTGCCAGGAGGTG

GAACCAAGACCCTTCAGCAGATAATCAATCAGTTGGAGTACCTGTCCCGTAAGGGAAAGCTGGAACTGCAGCGTTCAGCCCGGCAT

CTCGATATTCCCGTTCCGCCGGATCAAATCCGTGAGCTTGCCCAAAGCTGGGTTACGGAGGCCGGGATTTATGACGAAAGTCAGTC

AGACGATGATAGGCAACAAGACTTAACAACACACATTATTGTAAGCTTCCCCGCAGGTACCGACCAAACCGCAGCTTATGAAGCCA

GCCGGGAATGGGCAGCCGAGATGTTTGGGTCAGGATACGGGGGTGGCCGCTATAACTATCTGACAGCCTACCACGTCGACCGCGAT

CATCCACATTTACATGTCGTGGTCAATCGTCGGGAACTTCTGGGGCACGGGTGGCTGAAAATATCCAGGCGCCATCCCCAGCTGAA

TTATGACGGCTTACGGAAAAAGATGGCAGAGATTTCACTTCGTCACGGCATAGTCCTGGATGCGACTTCGCGAGCAGAAAGGGGAA

TAGCAGAGCGACCAATCACATATGCTGAACATCGCCGCCTTGAGCGGATGCAGGCTCAAAAGATTCAATTCGAAGATACAGATTTT

GATGAGACCTCGCCTGAGGAAGATCGTCGGGACCTCAGTCAATCGTTCGATCCATTTCGATCGGACCCATCTACCGGCGAACCGGA

CCGTGCAACCCGACATGACAAACAACCGCTTGAACAGCACGCCCGTTTCCAGGAGTCCGCCGGCTCCAGCATCAAAGCCGACGCAC

GGATCCGCGTATCATTGGAGAGCGAGCGGAGTGCCCAACCATCCGCGTCCAAAATCCCTGTAATTGGGCATTTCGGGATTGAGACT

TCCTATGTCGCTGAAGCCAGCGTGCGCAAACGAAGCGGCATTTTCGGTACTTCTCGCCCGGTGACTGACGTTGCCATGCACACAGT

CAAGCGCCAGCAGCGATCAAAACGACGTAATGACGAGGAGGCAGGTCCGAGCGGAGCAAACCGTAAAGGATTGAAGGCTGCGCAAG

TTGATTCCGAGGCAAATGTCGGTGAGCAAGACACTCGCGATGACAGCAACAAGGCGGCTGATCCGGTGTCTGCTTCCATCGGTACC

GAGCAACCGGAAGCTTCTCCAAAGCGTCCGCGTGACCGTCACGATGGAGAATTGGGTGGACGCAAACGTGCAAGAGGTAATCGTCG

CTCGAGCTCGAGCGGGGGGACCTAGAGACAGGAAGGACCGAATAATGGCCGCGG

SEQID37

ATGGCTTCTGTGCTGGCTTCTCTGTTTCCAAAACTGGGCTCTTTGGGTACTTCAGATCATGCTTCTGTTGTATCCATCAACCTCTT

TGTGGCACTCCTTTGTGCTTGCATCATCATTGGTCATCTCTTGGAGGAGAACCGCTGGGTTAATGAGTCCATTACTGCCCTCATAA

TTGGTTTGTGTACAGGAGTGGTTATCTTGCTCGTAAGTGGTGGAAAGAGCTCACACCTTCTGGTTTTCAGTGAAGATCTCTTTTTC

ATATATGTACTTCCTCCAATCATATTTAATGCAGGGTTTCAGGTAAAAAAGAAGCAATTTTTCGTAAACTTCATTACTATAATGAT

GTTCGGAGCCATTGGTACCCTGGTCTCATGTGCCATTATATCATTAGGTGCCATTCAAACTTTCAAGAAGTTGGACATTGAATTTC

TAGATATTGGGGATTATCTTGCAATTGGAGCAATATTTGCTGCCACAGATTCCGTCTGCACATTGCAGGTCCTACATCAGGATGAG

ACACCCCTCCTTTACAGTCTTGTATTTGGAGAAGGAGTTGTAAATGATGCTACATCGGTGGTGCTTTTCAATGCTATTCAAAACTT

CGACCTTACGAGCATGAATCCCAGTATAGCCCTCAGTTTCCTTGGCAACTTCTTCTATCTGTTCCTTGCTAGCACTTTACTGGGAG

CAGGAACTGGTCTTCTTAGTGCTTACATTATCAAGAAGCTATATTTTGGCAGGCACTCCACAGATCGTGAGGTTGCCCTTATGATG

CTCATGGCTTACTTATCATACTTGCTGGCCGAATTATTCTATTTGAGTGGGATTCTCACCGTCTTTTTCTGTGGTATTGTAATGTC

TCACTACACTTGGCACAATGTGACCGAGAGTTCAAGAGTCACTACAAGGCACACTTTTGCAACTTTGTCATTTCTTGCAGAGACTT

TCCTCTTCCTCTATGTCGGCATGGATGCTTTGGATATCGAGAAGTGGAAATTTGTTGGTGACAGGCCTGGATTATCAATTTCCGTG

AGTTCAATACTGATGGGACTAATCTTGCTTGGGAGAGCTGCCTTTGTTTTTCCATTATCATTCTTATCCAACTTAATGAAGAAATC

CTCGGAGCAAAAAATTACCTTTAGGCAGCAAGTGATAATATGGTGGGCAGGTTTGATGAGAGGCGCAGTGTCCATGGCACTGGCAT

ATAATAAGTTCACTCGTGGGGGACACACTCAACTGCAGGACAATGCAATAATGATTACCAGCACGATAACCATTGTTCTATTCAGC

ACAATGGTATTCGGTTTAATGACAAAACCCCTTATAAGTCTCCTGCTGCCACCACAGAGGCAATTGAGTACAGTGTCATCAGGCGC

AAATACTCCAAAGTCTCTAACAGCCCCACTCCTAGGCAGTCGAGAGGACTCTGAAGTTGATTTAAATGTTCCAGATCTTCCTCACC

CACCAAGTTTGAGGATGCTACTTACCGCACCAAGTCATAAAGTGCATCGGTACTGGCGCAAGTTTGACGATGCATTCATGCGCCCT

ATGTTTGGTGGTCGGGATTTGCTCCTCCTGCCCCTGGTTCTCCAACGGAACAGGGTCCTGAGGTACCAATC

SEQID38

TTGGTTTGTGTACAGGAGTGGTTATCTTGCTCGTAAGTGGTGGAAAGAACTCACACCTTCTGGTTTTCAGTGAAGATCTCTTTTTC

ATATATGTACTTCCTCCAATCATATTTAATGCAGGGTTTCAGGTAAAAAAGAAGCAATTTTTCGTGAACTTCATTACTATAATGAT

GTTCGGAGCCATTGGTACCCTGGTCTCATGTGCCATTATATCATTAGGTGCAATTCAAACTTTCAAGAAGTTGGACATTGAATTTC

TGACCTTACGAGCGTGAATCCCAGTATAGCCCTCAGTTTCCTTGGCAACTTCTTCTATCTGTTCCTTGCTAGCACTTTACTGGGAG

CAGGAACTGGTCTTCTTAGTGCTTACATTATCAAGAAGCTGTATTTTGGCAGGCACTCCACAGATCGTGAGGTTGCCCTTATGATG

CTCATGGCTTACTTATCATACATGCTGGCTGAACTATTCTATTTGAGTGGGATTCTCACTGTATTTTTCTGTGGTATTGTAATGTC

TCATTACACTTGGCACAATGTGACCGAGAGTTCAAGAGTCACTACAAGGCACGCTTTTGCAACTTTGTCATTTCTTGCAGAGACTT

AGTTCAATACTGATGGGATTAATCTTGCTGGGGAGAGCTGCCTTTGTTTTTCCATTATCATTCTTCTCCAACTTAATGAAGAAATC

ATAATAAGTTCACTCGTTGGGGGACACACTCAACTGCAGGACAATGCAATAATGATTACCAGCACGATAACCATTGTTCTATTCAGC

ACAATGGTATTCGGTTTAATGACAAAACCCCTTATAAGTCTCCTGCTGCCACCACAGAGGCAATTGAGTACAGTGTCATCAGGTGC

ATGTTTGGTGGTCGGGGATTTGCTCCTCCTGCCCCTGGTTCTCCAACGGAACAGGGTCCATGAGGTACAATC

SEQID39

ATGGAAAATTCGGTACCCAGGACTGTAGAAGAAGTATTCAACGATTTCAAAGGTCGTAGAGCTGGTTTAATCAAAGCACTAACTAC

AGATGTCGAGAAGTTTTATCAATCGTGTGATCCTGAAAAGGAGAACTTGTGTCTCTATGGGCTTCCTAATGAAACATGGGAAGTAA

ACCTCCCTGTAGAGGAGGTGCCTCCAGAACTTCCGGAGCCAGCATTGGGCATAAACTTCGCACGTGATGGAATGCAAGAGAAAGAC

TGGTTATCACTTGTTGCTGTTCACAGTGATTCATGGCTGCTTTCTGTTGCATTTTACTTTGGTGCAAGGTTTGGGTTCGGCAAGAG

TGAAAGGAAGAGGCTTTTCCAAATGATAAATGATCTCCCAACAGTGTTTGAAGTTGTTACCGGAGCTGCTAAACAGACACGTGATC

CCCCTCACAACAATAGCAACAAAAGCAAATCAAGTGGAAAGCCTCGACAGCCAGAGTCCCAACTCAAGGCAGTAAAGGTGTCTCCA

CCTAAAATGGAGAACGACAGTGGGGAGGAGGAAGAAGAAGAAGAGGATGAACAAGGAGCAACTCTCTGTGGAGCTTGTGGTGATAA

TTATGCCACTGATGAATTCTGGATTTGCTGTGATATTTGTGAGAGATGGTTCCATGGCAAATGTGTGAAGATTACCCCAGCAAAAG

CTGAGCATATCAAGCAGTACAAGTGTCCTAGTTGCAGTAGCAAGAGAGCTAGAGTTTAA

SEQID40

TGACATCTGCCAATAAAGCCAAGAATAATTGGCATTAACATGACCAAAAAAATGGTTTGGCAGCATTAAGTCAAATAAAAAAGCTA

CTTTAATATAAAATAATATTAAAATGCTTAATAACCAACAGTTTATAAGAAGGTTAATGTTAACATGGATGAGGAATGACCAAAAG

GGGAATTATATATTAACCTTTAAATCAATCTAATTCTCTCTTTTTGTTTCTAGCTATATTTACTCGATAGATAAACTCTCTTACTT

GACGAATTTTTTGATACAAGAAGACATATTTCATCATGATTTTAATTCGTCGTGTCAAATTTATTAAATAGTTTAATTTTAATCGT

AAATTTAGATATGAAATTTAAAAAAAAATAAATATATACATATTTGAAGAATACATAAAAAGTACATATAAATCACAAATATTTAA

TAATTCAAGATATTAAAACACATAGAAAAATAATTACTTACAAAGAAATTCTTATTTGAATCCTCTAAATTCGAGAAGTGCAACAC

AAACTGAGACGAAGAAAATGAATAATAATATTTGATAAGAAATTTATTATAATTGAATGACCATTTAAGTAATTACGGGTAATAAC

ACAATAAGGAACTGTAGTCATTTTTAATACATGGCAAGGAATATGAGAGTGTGATGAGTCTATAAATAGAAGGCTTCATTAGTGTA

GAGGAGTCACAAACAAGCAATACACAAATAAAATTAGTAGCTTAAACAAGATG

SEQID56

TTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTGTCTACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGG

TCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAA

AAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTC

AGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTC

SEQID94

TGGCAGGATATATGAGTGTGTAAAC

SEQID95

TTGGCAGGATATATCCCTCTGTAAAC

Claims

1. modify the method for selected plant characteristic, comprising:

A. the polynucleotide with expectation stably transform the cell of selected plant, and the polynucleotide of wherein said expectation constitute by selected plant, phase kindred plant or with the natural acid sequence of the interfertile plant of selected vegetalitas basically,

B. obtain the plant of stable conversion from described plant transformed cell, wherein said conversion plant contains the polynucleotide that stably are incorporated into the described expectation in the genome, and the polynucleotide of wherein said expectation are modified described characteristic.

2. according to the method for claim 1, further comprise the described vegetable cell of selected marker's cotransfection that is used in transient expression in the described vegetable cell, identify the plant transformed cell, from described plant transformed cell, obtain to transform plant, wherein integrate described selected marker astatically, and the polynucleotide of described expectation stably are incorporated in the genome.

3. the process of claim 1 wherein that described plant is a monocotyledons.

4. the method for claim 3, wherein said monocotyledons is selected from the group that is made of wheat, turf, turfgrass, cereal grass, corn, paddy rice, oat, wheat, barley, jowar, orchid, Herba Iridis japonicae, lily, onion, banana, sugarcane, jowar and palm.

5. the process of claim 1 wherein that described plant is a dicotyledons.

6. the method for claim 5, wherein said dicotyledons is selected from the group that is made of potato, tobacco, tomato, beet, asparagus broccoli, cassava, sweet potato, pepper, cotton, poinsettia, leguminous plants, clover, soybean, Radix Dauci Sativae, strawberry, lettuce, Oak Tree, maple, English walnut, rose, peppermint, pumpkin, daisy and Root and stem of Cholla.

7. the process of claim 1 wherein that described characteristic is from health and nutritive property by raising, the storage that improves, the output that improves, the enhanced salt tolerance, enhanced is to the tolerance of heavy metal, the tolerance that improves to arid, the tolerance that improves to disease, the tolerance that improves to insect, the tolerance that improves to water stress, enhanced is to the tolerance of cold-peace frost, the color that increases, the sweet taste that increases, the vigor that improves, the taste that improves, the structure of improving, the phosphate content that reduces, the percentage of germination that improves, the little nutrition that improves is taken in, the starch that improves is formed, select in the group that the life-span of the flower that improves constitutes.

According to the process of claim 1 wherein the polynucleotide of described expectation comprise the PPO gene, converting enzyme inhibitor gene, salt tolerance gene of P-DNA, GBSS promotor, Ubi7 promotor, Ubi3 promotor, PIP promotor, modification, the leader sequence relevant with R1, with Starch phosphorylase relevant leader sequence, with R1 relevant tailer sequence, tailer sequence, Ubi intron, GBSS transcribed spacer, the UbiT relevant with SBE.

9. the process of claim 1 wherein and transform described vegetable cell by agrobacterium-mediated being converted.

10. the method for claim 9, wherein agrobacterium-mediated conversion depend on uses at least one two carrier.

11. the method for claim 10, wherein agrobacterium-mediated method for transformation uses first pair of carrier and second pair of carrier.

12. the method for claim 11, wherein the first pair of carrier contains the polynucleotide of described expectation, and second pair of carrier contains functional selectable marker gene, wherein described functional selectable marker gene is operably connected on promotor and the terminator.

13. pass through the plant of the method preparation of claim 1.

14. modify the method for the characteristic of selected plant, comprising:

(a) identify the characteristic that to modify;

(b) make up basically by from described selected plant, phase kindred plant or with the interfertile plant of described selected vegetalitas first polynucleotide of constituting of isolating natural genetic elements, wherein said genetic elements can be modified the expression of the functional gene of the described characteristic of control;

(c) make up second polynucleotide that comprises functional selected marker;

(d) with the vegetable cell of described first and second described selected plant of polynucleotide cotransfection;

(e) the described functional selected marker's of selection transient expression;

(f) screening is with described first polynucleotide stable conversion but do not contain the vegetable cell that is incorporated into described second polynucleotide in the genome;

(g) plant of acquisition stable conversion from the described plant transformed cell that the expression process that shows described characteristic is modified.

15. the method for claim 14, wherein said plant is a monocotyledons.

16. the method for claim 15, wherein said monocotyledons is selected from the group that is made of wheat, turf, turfgrass, cereal grass, corn, paddy rice, oat, wheat, barley, jowar, orchid, Herba Iridis japonicae, lily, onion, banana, sugarcane, jowar and palm.

17. the method for claim 14, wherein said plant is a dicotyledons.

18. the method for claim 17, wherein said dicotyledons is selected from the group that is made of avocado, potato, tobacco, tomato, beet, asparagus broccoli, cassava, sweet potato, pepper, cotton, poinsettia, leguminous plants, clover, soybean, Radix Dauci Sativae, strawberry, lettuce, Oak Tree, maple, English walnut, rose, peppermint, pumpkin, daisy and Root and stem of Cholla.

19. the method for claim 14, wherein said characteristic is from health and nutritive property by raising, the storage that improves, the output that improves, the enhanced salt tolerance, enhanced is to the tolerance of heavy metal, the tolerance that improves to arid, the tolerance that improves to disease, the tolerance that improves to insect, the tolerance that improves to water stress, enhanced is to the tolerance of cold-peace frost, the color that increases, the sweet taste that increases, the vigor that improves, the taste that improves, the structure of improving, the phosphate content that reduces, the percentage of germination that improves, the little nutrition that improves is taken in, the starch that improves is formed, select in the group that the life-span of the flower that improves constitutes.

20. the method for claim 14, wherein said genetic elements comprise in promotor, interest sequence, terminator, enhanser, intron, transcribed spacer or the regulatory element at least one.

21. the method for claim 14 wherein transforms described vegetable cell by agrobacterium-mediated being converted.

22. depending on, the method for claim 21, wherein agrobacterium-mediated conversion use at least one two carrier.

23. the method for claim 22, wherein agrobacterium-mediated method for transformation uses first pair of carrier and second pair of carrier.

24. the method for claim 23, wherein first pair of carrier has described first polynucleotide, and second pair of carrier has described second polynucleotide.

25. modify the method for selected plant function genetic expression, comprising:

(a) make up basically by from described selected plant, with described selected plant plant mutually of the same race or with the interfertile plant of described selected vegetalitas first polynucleotide that constitute of isolating natural genetic elements, wherein said natural genetic elements can be modified the expression of described functional gene;

(b) make up second polynucleotide that comprise the selected marker who is operably connected on promotor and the terminator;

(c) with the vegetable cell of the described selected plant of the described first and second polynucleotide cotransfections;

(d) the described selected marker's of selection transient expression;

(e) screening stably transforms with described first polynucleotide but does not contain the vegetable cell that is incorporated into second polynucleotide in the genome; And

(f) from showing that described expression of gene is through obtaining plant transformed the described plant transformed cell of modifying.

26. the method for claim 25, wherein said plant is a monocotyledons.

27. the method for claim 26, wherein said monocotyledons is selected from the group that is made of wheat, turf, turfgrass, cereal grass, corn, paddy rice, oat, wheat, barley, jowar, orchid, Herba Iridis japonicae, lily, onion, banana, sugarcane, jowar and palm.

28. the method for claim 25, wherein said plant is a dicotyledons.

29. the method for claim 28, wherein said dicotyledons is selected from the group that is made of potato, tobacco, tomato, beet, asparagus broccoli, cassava, sweet potato, pepper, cotton, poinsettia, leguminous plants, clover, soybean, Radix Dauci Sativae, strawberry, lettuce, Oak Tree, maple, English walnut, rose, peppermint, pumpkin, daisy and Root and stem of Cholla.

30. the method for claim 25 wherein transforms described vegetable cell by agrobacterium-mediated being converted.

31. depending on, the method for claim 30, wherein agrobacterium-mediated conversion use at least one two carrier.

32. the method for claim 31, wherein agrobacterium-mediated method for transformation uses first pair of carrier and second pair of carrier.

33. the method for claim 32, wherein first pair of carrier has described first polynucleotide, and second pair of carrier has described second polynucleotide.

34. the method for claim 25, wherein said first polynucleotide comprise the PPO gene of P-DNA, GBSS promotor, Ubi7 promotor, Ubi3 promotor, PIP promotor, modification, the tailer sequence relevant with PPO, converting enzyme inhibitor gene, salt tolerance gene, with R1 relevant leader sequence, with Starch phosphorylase relevant leader sequence, with R1 relevant tailer sequence, with SBE among relevant tailer sequence, Ubi intron, GBSS transcribed spacer, the UbiT at least one.

35. the method for claim 25, wherein said second polynucleotide comprise in virD2 polynucleotide, codA polynucleotide and the codA::upp fusion polynucleotides of marker gene optionally, Ω sudden change at least one.

36. pass through the plant of the method preparation of claim 25.

37. show and compare with the non-transgenic plant in its source, the transgenic plant of the expression of characteristic through modifying, wherein the polynucleotide with the expectation that is made of natural genetic elements basically stably transform described transgenic plant, these polynucleotide be from described plant, plant mutually of the same race or with the interfertile plant of described vegetalitas separate, wherein said polynucleotide are modified the expression of described characteristic.

38. according to the plant of claim 37, wherein said plant is a monocotyledons.

39. according to the plant of claim 38, wherein said monocotyledons is selected from the group that is made of wheat, turf, turfgrass, cereal grass, corn, paddy rice, oat, wheat, barley, jowar, orchid, Herba Iridis japonicae, lily, onion, banana, sugarcane, jowar and palm.

40. according to the plant of claim 37, wherein said plant is a dicotyledons.

41. according to the plant of claim 40, wherein said dicotyledons is selected from the group that is made of potato, tobacco, tomato, beet, asparagus broccoli, cassava, sweet potato, pepper, cotton, poinsettia, leguminous plants, clover, soybean, Radix Dauci Sativae, strawberry, lettuce, Oak Tree, maple, English walnut, rose, peppermint, pumpkin, daisy and Root and stem of Cholla.

42. according to the characteristic of claim 37, wherein said characteristic is from health and nutritive property by raising, the storage that improves, the output that improves, the enhanced salt tolerance, enhanced is to the tolerance of heavy metal, the tolerance that improves to arid, the tolerance that improves to disease, the tolerance that improves to insect, the tolerance that improves to water stress, enhanced is to the tolerance of cold-peace frost, the color that increases, the sweet taste that increases, the vigor that improves, the taste that improves, the structure of improving, the phosphate content that reduces, the percentage of germination that improves, the little nutrition that improves is taken in, the starch that improves is formed, select in the group that the life-span of the flower that improves constitutes.

43. according to the polynucleotide of the expectation of claim 37, wherein said polynucleotide comprise the PPO gene of P-DNA, GBSS promotor, Ubi7 promotor, Ubi3 promotor, PIP promotor, modification, the tailer sequence relevant with PPO, converting enzyme inhibitor gene, salt tolerance gene, with R1 relevant leader sequence, with Starch phosphorylase relevant leader sequence, with R1 relevant tailer sequence, with SBE among relevant tailer sequence, Ubi intron, GBSS transcribed spacer, the UbiT at least one.

44. an isolating edge sample nucleotide sequence, its size is 20-100bp, with the T-DNA edge sequence of agrobacterium tumefaciens 52% to 96% sequence identity is arranged.

45. the isolating nucleotide sequence of claim 44 wherein separates described nucleotide sequence from monocotyledons.

46. the isolating Nucleotide of claim 45, wherein said monocotyledons is selected from the group that is made of wheat, turf, turfgrass, cereal grass, corn, paddy rice, oat, wheat, barley, jowar, orchid, Herba Iridis japonicae, lily, onion, banana, sugarcane, jowar and palm.

47. the isolating nucleotide sequence of claim 44 wherein separates described nucleotide sequence from dicotyledons.

48. the isolating nucleotide sequence of claim 47, wherein said dicotyledons is selected from the group that is made of potato, tobacco, tomato, beet, asparagus broccoli, cassava, sweet potato, pepper, cotton, poinsettia, leguminous plants, clover, soybean, Radix Dauci Sativae, strawberry, lettuce, Oak Tree, maple, English walnut, rose, peppermint, pumpkin, daisy and Root and stem of Cholla.

49. the nucleotide sequence of claim 44, it separates from potato and has at the nucleotide sequence shown in SEQ IDNO.54 or 55.

50. the nucleotide sequence of claim 44, it separates from wheat and has at the nucleotide sequence shown in SEQ IDNO.94 or 95.

51. the preparation method of the plant of the polynucleotide stable conversion of expecting comprises:

(a) separating both sides from described plant is P-DNA of edge sample sequence, and the T-DNA edge sequence of wherein said edge sample sequence and agrobacterium tumefaciens has 52% to 96% sequence identity.

(b) between described P-DNA edge sample sequence, insert the polynucleotide of described expectation to form the P-DNA construction; With

(c) transform the vegetable cell of described plant with described P-DNA construction; With

(d) from described plant transformed cell, regain plant with described P-DNA construction stable conversion.

52. according to the method for claim 51, wherein described P-DNA construction is loaded on the carrier that is made of the main chain integration marker gene, selects not contain the plant transformed cell of described main chain integration marker gene.

53. according to the method for claim 52, wherein said main chain integration marker gene is selected from the group that is made of the cell fission plain gene, does not wherein select to show the plants shoots of excessive production phytokinin phenotype.

54. according to the method for claim 53, wherein said main chain integration marker gene is the IPT gene, and does not select the display abnormality phenotype maybe can not grow the plants shoots of root.

55. according to the method for claim 52, wherein said vegetable cell is from monocotyledons.

56. the method for claim 55, wherein said monocotyledons is selected from the group that is made of wheat, turf, turfgrass, cereal grass, corn, paddy rice, oat, wheat, barley, jowar, orchid, Herba Iridis japonicae, lily, onion, banana, sugarcane, jowar and palm.

57. the method for claim 52, wherein said vegetable cell is from dicotyledons.

58. the method for claim 57, wherein said dicotyledons is selected from the group that is made of avocado, potato, tobacco, tomato, beet, asparagus broccoli, cassava, sweet potato, pepper, cotton, poinsettia, leguminous plants, clover, soybean, Radix Dauci Sativae, strawberry, lettuce, Oak Tree, maple, English walnut, rose, peppermint, pumpkin, daisy and Root and stem of Cholla.

59. the method for claim 51 is wherein by the agrobacterium-mediated described vegetable cell of the transfection that is converted.

60. depending on, the method for claim 59, wherein agrobacterium-mediated conversion use at least a pair of carrier.

61. the method for claim 60, wherein agrobacterium-mediated method for transformation uses first pair of carrier and second pair of carrier.

62. the method for claim 61, wherein first pair of carrier has described P-DNA construction.

63. the method for claim 61, wherein the second pair of carrier comprises at least one in the virD2 gene of negative selected marker and Ω sudden change, wherein negative selected marker is positioned at the right hand edge sequence of T-DNA and the left hand edge sequence of T-DNA, and wherein the virD2 gene of Ω sudden change is positioned at the main chain of second pair of carrier.

64. comprise the carrier of the nucleotide sequence of claim 44.

65. 5 '-on 3 '-direction, basically the P-DNA that is made of a T-DNA edge sample sequence, promotor, the polynucleotide sequence that is operably connected to the expectation on the promotor, terminator and the 2nd T-DNA edge sample sequence, wherein edge sample sequence and T-DNA edge sequence have and are lower than 100% sequence identity.

66. the P-DNA of claim 65, the polynucleotide of wherein said T-DNA edge sample sequence, described promotor, described expectation and described terminator separate from identical plant, identical plant species or the interfertile plant of property.

67. the P-DNA of claim 65, it further is made of the selected marker basically.

68. the P-DNA of claim 67, wherein said T-DNA edge sample sequence, described promotor, described expectation polynucleotide, described terminator and described selected marker separate from identical plant, identical plant species or the interfertile plant of property.

69. the P-DNA of claim 65, wherein Qi Wang polynucleotide sequence is the upstream or the downstream sequence of gene coding region, and wherein upstream sequence is a leader sequence, and wherein downstream sequence is a tailer sequence.

70. the P-DNA of claim 69, wherein said T-DNA edge sample sequence, described promotor, described leader sequence, described tailer sequence, described terminator and described selected marker separate from identical plant, identical plant species or the interfertile plant of property.

71. isolating nucleotide sequence that comprises isolating GBSS promotor from potato.

72. the isolating nucleotide sequence of claim 71, it has the nucleotide sequence of SEQ ID NO.6 or 13.

73. comprise the carrier of the P-DNA of claim 65.

74. according to the carrier of claim 74, wherein said promotor is adjustable promotor.

75. according to the carrier of claim 74, wherein adjustable promotor is to temperature sensitive.

76. according to the carrier of claim 75, wherein adjustable promotor is a wheat wcs120 promotor.

77. according to the carrier of claim 74, wherein said promotor is under sequential is regulated.

78. according to the carrier of claim 77, wherein said promotor is the carboxylase promotor.

79. according to the carrier of claim 78, wherein the carboxylase promotor is a corn carboxylase promotor.

80. according to the carrier of claim 74, wherein by any adjusting promotor in dormin, wound, methyl jasmonate or the gibberic acid.

81. 0 carrier according to Claim 8, wherein said promotor is the promotor of selecting from the promotor of the promotor of the promotor of Rab 16A gene, αDian Fenmei gene or pin2 gene.

82. according to the carrier of claim 73, wherein said promotor is tissue-specific promotor.

83. according to the carrier of claim 69, wherein said leader sequence is the part of the 5 '-non-translational region relevant with the endogenous gene of selected plant species cell.

84. carrier according to claim 69, wherein 5 '-non-translational region is the upstream sequence of gene start codon, this gene is from by the PPO gene, the R1 gene, the HOS1 gene, the high half Guang hydrolase gene of S-adenosine, II class styracin-4-'-hydroxylase gene, cinnyl-CoA reductase gene, the cinnyl alcohol dehydrogenase gene, caffeoyl coenzyme A O-methyl transferase gene, the actin depolymerizing factor gene, the Nin88 gene, Lol p5 gene, the allergen gene, the P450 '-hydroxylase gene, ADP-glucose pyrophosphorylase gene, the proline dehydrogenase gene, in-1,4-beta-glucanase gene, zeaxanthin epoxidase gene, and select in the group of 1-Aminocyclopropane-1-carboxylate synthase gene formation.

85. carrier according to claim 69, wherein said tailer sequence is the part with 3 '-non-translational region of the gene of the gene-correlation that is positioned at the terminator codon downstream that is selected from following gene: the PPO gene, the R1 gene, the HOS1 gene, the high half Guang hydrolase gene of S-adenosine, II class styracin-4-'-hydroxylase gene, cinnyl-CoA reductase gene, the cinnyl alcohol dehydrogenase gene, caffeoyl coenzyme A O-methyl transferase gene, the actin depolymerizing factor gene, the Nin88 gene, Lol p5 gene, the allergen gene, the P450 '-hydroxylase gene, ADP-glucose pyrophosphorylase gene, the proline dehydrogenase gene, in-1,4-beta-glucanase gene, zeaxanthin epoxidase gene, and 1-Aminocyclopropane-1-carboxylate synthase gene.

86. according to the carrier of claim 65, it further comprises the transcribed spacer element as Ubi intron sequences or GBSS transcribed spacer sequence.

87. according to the carrier of claim 65, wherein said terminator is 3 '-non-translational region of Ubi3 terminator sequence or endogenic plant gene.

88. carrier according to claim 65, it comprises further and is operably connected to the selected marker on the constitutive promoter and is operably connected to Cre gene on the inducible promoter that wherein the both sides of selected marker and Cre gene are the recognition site of first recombinase and the recognition site of second recombinase.

89. 8 carrier according to Claim 8, wherein the recognition site of the recognition site of first recombinase and second recombinase is the 1ox site.

90. 8 carrier according to Claim 8, but wherein said inducible promoter is the promotor or the sequential promotor of temperature sensitive promotor chemical induction.

91. 8 carrier according to Claim 8, wherein inducible promoter is Ha hsp17.7G4 promotor, wheat wcs120 promotor, Rab 16a gene promoter, αDian Fenmei gene promoter, pin2 gene promoter, carboxylase promotor.

92. according to the carrier of claim 65, it further comprises the marker gene of plant origin.

93. according to the carrier of claim 92, wherein the marker gene of plant origin is enol pyruvoyl-3-phosphoric acid shikimic acid synthase gene, salt tolerance gene or PST1 gene, PST2 gene or PST3 gene.

94. the method for modified plant cell, comprise the P-DNA sequence is inserted in the genome of vegetable cell, wherein on 5 ' to 3 ' direction, P-DNA is basically by first T-DNA edge sample sequence, promotor, be operably connected to the polynucleotide sequence of the expectation on the promotor, terminator, and second T-DNA edge sample sequence constitutes, wherein edge sample sequence and T-DNA edge sequence have and are less than 100% sequence identity, T-DNA edge sample sequence wherein, promotor, the polynucleotide of expectation, and terminator all separates certainly or belongs to the genome of vegetable cell, wherein Qi Wang polynucleotide comprise the leader sequence relevant with the non-coding region in the upstream of plant gene or downstream or the justice and the antisense sequences of tailer sequence, wherein the expression of Qi Wang polynucleotide produces the double-stranded RNA transcript of the target gene relevant with the polynucleotide of expectation, thus the modified plant cell.

95. the method for modified plant comprises:

(i) at least one cell of the carrier transfection plant of usefulness claim 88;

(ii) select to express the cell of selected marker;

(iii) separate the cell of expressing selected marker;

(iii) in isolated cells, induce the Cre expression of gene;

The (iv) cell of culture of isolated; With

(ii) observe the phenotype of culturing cell;

The phenotype that wherein is different from the vegetable cell of untransfected shows that the target vegetable cell is modified.

96., wherein be tested and appraised which cell resistance microbiotic and finish the selection step according to the method for claim 14 or 95.

97. the identified gene group contains the method for the target vegetable cell of P-DNA, comprise with the carrier of claim 65 and the carrier cotransfection plant target cell that derives from second edaphic bacillus, the carrier that wherein derives from second edaphic bacillus comprises that both sides are the marker gene of T-DNA edge sequence or T-DNA edge sample sequence and the virD2 gene of Ω sudden change, wherein the P-DNA with the carrier of claim 65 is incorporated in the genome of plant target cell, and any part that wherein will not derive from the carrier of second edaphic bacillus is incorporated in the genome of plant target cell, and wherein the virD2 gene of Ω sudden change is positioned at main chain.

98. according to the method for claim 97, the mark that wherein derives from the carrier of second edaphic bacillus is a neomycin phosphotransferase gene.

99. the identified gene group contains the method for target vegetable cell of at least a portion of the integration box of with good grounds claim 98, it further comprises the cell that is chosen in sequential growth survival in the substratum that contains kantlex, and wherein the genome of selected cell only contains the integration box.

100. according to any one method in the claim 94,95 or 97, its vegetable cell that hits is positioned at plant.

101. comprise the plant of cell that at least one its genome contains the P-DNA of with good grounds claim 65.

102. comprise at least one its genome by the plant of manual maneuvering with the cell that only contains plant origin nucleic acid, wherein the none cell contains the exogenous nucleic acid in the genome that is integrated into cell.

103. the plant of claim 102, wherein said cell can be expressed in the nucleic acid of plant origin at least one, this expression of nucleic acids has been modified the characteristic relevant with plant.

104. reduce the abrasive method of blackspot in the selected plant species, comprise that (i) will be incorporated into the P-DNA that edge sample sequence is described in the genome of selected plant species, wherein P-DNA only comprises the natural polynucleotide from selected plant species of belonging to or separate, perhaps comprise natural belong to or separate from the polynucleotide of the affine plant species of selected plant species, wherein P-DNA 5 '-on 3 '-direction basically by promotor, the leading nucleotide sequence of the just direction of PPO gene, the antisense orientation sequence of leading nucleotide sequence and terminator sequence constitute, wherein said promotor produces double stranded rna molecule, wherein said double stranded rna molecule causes endogenous PPO genetic expression to reduce, and therefore reduces the blackspot scratch in the plant.

105. reduce the abrasive method of blackspot in the selected plant species, comprise and to be incorporated into the P-DNA that edge sample sequence is described in the genome of selected plant species, wherein P-DNA only comprises the natural nucleotide sequence from selected plant species of belonging to or separate, perhaps comprise natural belong to or separate from the polynucleotide of the affine plant species of selected plant species, wherein P-DNA 5 '-on 3 '-direction basically by promotor, the just direction of PPO gene trail nucleotide sequence, antisense orientation sequence and the terminator sequence of trailing nucleotide sequence constitute, wherein said promotor produces double stranded rna molecule, wherein said double stranded rna molecule causes PPO genetic expression to reduce, and therefore reduces the blackspot scratch in the plant.

106. reduce the cold method that sweetens of inducing in the selected plant species, comprise and to be incorporated into the P-DNA that edge sample sequence is described in the genome of selected plant species, wherein P-DNA only comprises isolating nucleotide sequence from selected plant species, perhaps comprise natural belong to or separate from the polynucleotide of the affine plant species of selected plant species, wherein P-DNA 5 '-on 3 '-direction basically by promotor, leading nucleotide sequence with the just direction of R1 gene-correlation, the antisense orientation sequence of leading nucleotide sequence and terminator sequence constitute, wherein said promotor produces double stranded rna molecule, wherein said double stranded rna molecule reduces the R1 expression of gene, reduces therefore that cold inductive sweetens in the plant.

107. reduce the cold method that sweetens of inducing in the selected plant species, comprise and to be incorporated into the P-DNA that edge sample sequence is described in the genome of selected plant species, wherein P-DNA only comprises isolating nucleotide sequence from selected plant species, perhaps by natural belong to or separate from the polynucleotide of the affine plant species of selected plant species, wherein P-DNA 5 '-on 3 '-direction basically by promotor, the nucleotide sequence of trailing with the just direction of R1 gene-correlation, antisense orientation sequence and the terminator sequence of trailing nucleotide sequence constitute, wherein said promotor produces double stranded rna molecule, wherein said double stranded rna molecule reduces the R1 expression of gene, reduces therefore that cold inductive sweetens in the plant.

108. the method for claim 8, wherein the interest sequence is a gene.

109. the method for claim 108, wherein said gene are through the polyphenoloxidase of modifying, polyphenol oxidase genes or R1 gene or R1 gene through modifying.

110. the method for claim 8, wherein said promotor is an inducible promoter.

111. the method for claim 8, wherein said terminator are the terminator sequences of yeast ADH.

112. the method for claim 8, wherein the interest sequence is leading or tailer sequence, and wherein leading or tailer sequence is represented the upstream or the downstream sequence of the natural gene of vegetable cell.

113. the method for claim 112, wherein the interest sequence comprises the leader sequence that is operably connected to the just direction on the antisense leader sequence.

114. the method for claim 112, wherein the interest sequence comprises the tailer sequence that is operably connected to the just direction on the antisense tailer sequence.

115. the method for claim 113, wherein the leader sequence construction 5 '-on 3 '-direction, comprise the leader sequence of promotor, just direction, the antisense sequences and the terminator of leader sequence, wherein the expression of leader sequence construction produces and is convenient to reduce the double stranded rna molecule of relative genetic expression.

116. the method for claim 115, wherein leader sequence is relevant with the coding region of PPO gene, R1 gene, L type phosphorylase gene or alpha-glucan phosphorylase gene, and is positioned at its upstream.

117. the method for claim 113, wherein the tailer sequence construction 5 '-on 3 '-direction, comprise the tailer sequence of promotor, just direction, the antisense sequences and the terminator of tailer sequence, wherein the expression of tailer sequence construction produces and is convenient to reduce the double stranded rna molecule of relative genetic expression.

118. the method for claim 117, wherein tailer sequence is relevant with the coding region of PPO gene, R1 gene, L type phosphorylase gene or alpha-glucan phosphorylase gene, and is positioned at its downstream.

119. the method for claim 9, it further comprises vegetable cell is exposed to second carrier that contains identification element, and wherein this is marked at that the of short duration face of land reaches in the plant transformed, and is not incorporated in the genome that transforms plant with being stabilized.

120. the method for claim 119, wherein said mark are the resistant genes of weedicide.

121. the method for claim 120, wherein said cell fission plain gene is antibiotic resistant gene.

122. the method for claim 119, wherein said mark is NPTII.

123. comprise the polynucleotide of the polynucleotide sequence of SEQ ID NO.93, wherein the length of these polynucleotide is 20 to 80 Nucleotide.

124. the polynucleotide of claim 123, the length of wherein said polynucleotide are 21 to 70 Nucleotide.

125. the polynucleotide of claim 123, the length of wherein said polynucleotide are 22 to 50 Nucleotide.

126. the polynucleotide of claim 123, wherein said polynucleotide length are 23 to 40 Nucleotide.

127. the polynucleotide of claim 123, wherein said polynucleotide length are 24 to 30 Nucleotide.

128. the method for claim 4, wherein before with second polynucleotide transfection with first polynucleotide transfection of plant cells.

129. the specific promotor of stem tuber shown in SEQ ID NO.40.

130. based on the method for preparing transgenic plant cells of edaphic bacillus, this commentaries on classics is incorporated into the selectable marker gene among the nuclear DNA with being stabilized because of vegetable cell does not contain, this method comprises:

A. make up first pair of carrier comprising basically the polynucleotide that the gene by described expectation constitutes, wherein this gene is operably connected on T-DNA edge sequence or the T-DNA edge sample sequence at its 5 ' and 3 ' end;

B. make up second pair of carrier that 5 ' and the 3 ' end that is included in described selected marker is operably connected to the selected marker on T-DNA edge sequence or the T-DNA edge sample sequence;

C. vegetable cell is cultivated with following material:

I. have described first and the edaphic bacillus bacterial strain of described second pair of carrier; Or

Ii. second edaphic bacillus (Agrobacterium) bacterial strain that has the first edaphic bacillus bacterial strain of described first pair of carrier and have described second pair of carrier;

D. after cultivating reasonable time on the substratum that contains appropriate selection reagent, selection is wherein examined described expectation gene integration among the DNA to plant, and described selected marker is not incorporated into the vegetable cell among the plant nuclear DNA.

131. according to the method for claim 130, wherein said selected marker is the resistant gene or the antibiotic resistant gene of weedicide.

132. according to the method for claim 131, wherein said antibiotic resistant gene is the nptII gene.

133. according to the method for claim 132, wherein said antibiotic resistant gene is the nptII structure gene that is operably connected on the terminator of the promotor of ubiquitin-7 gene and Alcohol Dehydrogenase from Yeast 1 (ADH) gene.

134. according to the method for claim 130, wherein at first described vegetable cell is cultivated with the described first edaphic bacillus bacterial strain, then subsequently described vegetable cell is cultivated with the described second edaphic bacillus bacterial strain, perhaps conversely.

135. according to the method for claim 130, wherein said first pair of carrier further comprises two integration marker genes, it can be used for detecting the vegetable cell with two carrier main chain sequence stable integrations.

136. according to the method for claim 135, wherein said pair of vector integration marker gene selected from the group that is made of herbicide resistance gene, antibiotics resistance gene or NPTII.

137. method according to claim 130, wherein said second pair of carrier further is included in the genetic fusant between bacterium Isocytosine deaminase (codA) and uracil phosphoribosyl transferase (upp) gene, this syzygy is inserted between T-DNA or T-DNA edge sample sequence, after the described first and second edaphic bacillus bacterial strains are cultivated, with vegetable cell be exposed to the 5-cytosine(Cyt) with select anti-those with described second pair of carrier plant transformed cell.

138. according to the method for claim 130, wherein said second pair of carrier further comprises the virD2 gene of Ω sudden change, the virD2 gene of wherein said Ω sudden change reduces the frequency among the nuclear DNA that described selected marker is incorporated into described plant.

139. the isolating edge sample nucleotide sequence of claim 44, wherein the length of this sequence is 25 Nucleotide.

140. the method for claim 61, wherein second pair of carrier comprises the selected marker of feminine gender and at least one that hinders in the gene of integrating, wherein Yin Xing selected marker is positioned at T-DNA right hand edge sequence and T-DNA left hand edge sequence, wherein hinders the gene of integrating to be positioned at the main chain of second pair of carrier.

141. the method for claim 99, wherein the sequential growth is 1 to 5 day.