CN1317051A - HMP-P rinase and TMP-PPase from arabidopsis thaliana and their use in herbicide screening - Google Patents

Abstract

The present invention discloses methods to screen chemicals for herbicidal activity using recombinantly produced enzymes having HMP-P kinase activity or TMP-PPase activity, and the use of thereby to identify herbicidal chemicals to suppress the growth of undesired vegetation. Furthermore, the present invention provides methods for the development of herbicide tolerance in plants, plant tissues, plant seeds, and plant cells using genes encoding enzymes having HMP-P kinase activity or TMP-PPase activity, and methods of using such transgenic plants to selectively suppress weed growth in crop fields.

Description

Arabidopis thaliana HMP-P kinases and TMP-PP enzyme and the purposes in herbicide screening thereof

The present invention relates to screen the method for the herbicidal compound that suppresses following enzymic activity, 4-methyl-5-(2-phosphoric acid ethyl) thiazole 2-methyl-4-aminopyrimidine-5-methyne transferring enzyme) or the TMP-PP enzyme described enzyme is that (2-methyl-4-amino-5-(methylol) pyrimidine bisphosphate:, from the beginning these two kinds of enzymes are all having enzymatic activity in the VitB1 biosynthesizing to the single phosphoric acid pyrophosphorylase of 2-methyl-4-amino-5-oxymethylpyrimidine monophosphate kinase (HMP-P kinases) or VitB1.The purposes of vegetation (vegetation) growth that the herbicidal compound control that the present invention relates to identify is not thus expected.The present invention also can be applicable to the exploitation of herbicide resistant plants, plant tissue, plant seed and vegetable cell.

The purposes that weedicide is used for controlling the vegetation of not expecting (as the weeds in crop field) is very general.Annual weedicide is sold above 15,000,000,000 dollars.Although use extensively like this, weeds control is still important and expensive problem to the peasant.

Effectively utilize weedicide to need a large amount of management work.For example, the stage of the time of application and method and ruderal plant growth is most important for obtain good effect with weedicide.Because multiple weeds kind has resistance to weedicide, preparation has effective herbicide to become to become more and more important.Can utilize the high-throughput screening method of taking recombinant DNA technology to find novel herbicide now.But recombinant production is to plant-growth and grow crucial metabolic enzymes, and is used as the weedicide target of screening novel enzyme activity inhibitor.The novel inhibitors of finding by such screening can be used as the weedicide of the vegetation growth that control do not expect.

Unfortunately, in soil, show the weeds spectrum agent of higher effectiveness, broad and degrade faster that weedicide also has bigger crop plants toxicity.A kind of method of head it off is to cultivate the crop of anti-or herbicide-tolerant.The crop hybrid kind of herbicide-tolerant or mutation can allow to use weedicide to kill weeds and the risk of harmless crop.The formation of tolerance makes can use weedicide to crop, and before this owing to the sensitivity of crop to weedicide, use (for example just using) can not be used or limit to this weedicide before emergency situation.For example, United States Patent (USP) 4761373 (Aderson etc.) relates to the plant that multiple imidazolone or sulphonamide herbicides is had resistance.Described resistance is given by the acetohydroxy acid synthase (AHAS) that changes.United States Patent (USP) 4975374 (Goodman etc.) relates to vegetable cell and the plant that contains encoding mutant glutamine synthetase (GS) gene, and it can resist the weedicide of known inhibition GS, as phosphinothricin and methionine(Met) sulfoximine.United States Patent (USP) 5013659 (Bedbrook etc.) relates to the plant of expressing mutant acetolactate synthase, and this enzyme is given the inhibition of plant opposing sulphonyl urine classes of herbicides.United States Patent (USP) 5162602 (Somers etc.) discloses the plant of tolerance cyclohexanedione herbicide class and aryloxy phenoxy propionic acid classes of herbicides.Give tolerance by the acetyl-CoA carboxylase (ACCase) that changes.For the purpose of being defined as clearly, some term definitions that are used for this specification sheets are as follows:

But activated dna sequence: the dna sequence dna that regulatory gene is expressed in genome especially Plant Genome.But the endogenous target gene complementation of activated dna sequence and genome.When but the activated dna sequence imported and expresses in cell, it suppressed target gene expression.But can comprise those codings with the activated dna molecule that the present invention unites use or play the molecule of dominance inhibitor effect, as translating or untranslated has adopted sequence, it can destroy gene function in the plant of stable conversion, with one or more plant normal growths of positive identification and the essential gene of growth.But a kind of preferred activated dna sequence is an antisense dna sequence.Target gene a kind of protein of preferably encoding is as biosynthetic enzyme, acceptor, signal transducer matter, structure gene product or plant-growth or survive translocator matter essential.In a preferred embodiment, target gene coding enzyme with HMP-P kinase activity or TMP-PP enzymic activity.The results of interaction of antisense sequences and target gene causes expression of target gene to be suppressed basically, thus kill plants, or suppress normal plants growth or growth at least.

But activated dna construct: a kind of recombinant DNA construction body, but comprise a kind of and activated dna sequence and effectively be connected the ground synthetic promoter, when its transfered cell (desirably, vegetable cell) do not express in the time of in, that is: for silence, unless exist a kind of can in conjunction with and activate the complete heterozygosis transcription factor of this synthetic promoter.But with activable DNA construct transfered cell, tissue or plant to form the stable transgenic strain can express the activated dna sequence.

Cofactor: needed natural response thing in the enzymic catalytic reaction, as organic molecule or metal ion.Cofactor is for example NAD (P), riboflavin (comprising FAD and FMN), folic acid, molybdoprotein (molybdopterin), VitB1, vitamin H, Thioctic Acid, pantothenic acid and coenzyme A, S-adenosylmethionine, pyridoxal phosphate, ubiquinone, methyl naphthoquinone.Randomly cofactor is renewable uses with huge profit.

Coupling is synthetic: a kind of enzymatic living beings is synthetic, wherein end product is synthetic by two or more order enzymatic steps, wherein the substrate that is used for first enzymatic step in one or more branches of bio-chemical pathway is added into reaction mixture, by first enzymatic conversion is intermediate product, and this intermediate product is become end product and need not to add intermediate product by second enzyme or a plurality of enzymatic conversion.

" chimeric " is used to represent a kind of dna sequence dna such as carrier or gene, its above dna sequence dna by the different sources that merges mutually by recombinant DNA technology constitutes, obtain that a kind of non-natural exists, especially be not present in dna sequence dna in the plant to be transformed.

" DNA reorganization. " DNA reorganization is the method that imports (preferably randomly) sudden change or reset in dna molecular, perhaps preferably produces the dna sequence dna exchange between two or more dna moleculars randomly.The dna molecular that derives from DNA reorganization is such reorganization dna molecular, and it is the dna molecular from the non-natural existence of at least one template DNA molecule.The reorganization dna encoding is a kind of relatively by the modified enzyme of enzyme of template DNA coding, preferably has the biologic activity of change for the enzyme of template DNA coding.

Enzymic activity: be meant that herein substrate for enzymatic activity is converted into the ability of product.A kind of substrate of enzyme comprises the natural substrate of enzyme, also comprises the analogue of natural substrate, and it can be product or product analogue by enzymatic conversion also.The active measurement of enzyme is by for example, in certain hour amount of product in the assaying reaction after the stage, or by being determined at the amount of residue substrate in the certain hour stage afterreaction mixture.Also can be by measuring the amount of the cofactor that remaining reaction is not used in the certain hour stage afterreaction mixture, or the method for the amount by being determined at the cofactor that uses in the certain hour stage afterreaction mixture is measured enzymic activity.Also can be by being determined at the amount (ATP that remains free energy in the certain hour stage afterreaction mixture or be rich in the kinetomeres donor, phosphoenolpyruvic acid, acetylphosphate or phosphocreatine), or by being determined at the free energy that uses in the certain hour stage afterreaction mixture or being rich in the amount (ADP of kinetomeres donor, pyruvic acid, acetate or creatine) measure the activity of enzyme.

Expression is meant in the plant native gene or genetically modifiedly transcribes and/or translate.In antisense constructs, for example, expression can only refer to transcribing of antisense DNA.

Gene is meant encoding sequence and relevant adjusting sequence, and wherein encoding sequence is transcribed into RNA, as mRNA, rRNA,, tRNA, snRNA or sense-rna.The example of regulating sequence is a promoter sequence, 5 ' and 3 ' non-translated sequence and terminator sequence.Other element that can exist is an intron for example.

Weedicide: a kind of chemical substance that is used to kill or suppress the growth of plant, vegetable cell, plant seed or plant tissue.

Allogeneic dna sequence: a kind of dna sequence dna, it is natural not relevant with the host cell that will import this dna sequence dna, comprises the multiple copy of the intrinsic dna sequence dna that non-natural exists.

Homologous DNA sequence: a kind of dna sequence dna, it is natural relevant with host cell to be imported.

Inhibition: the chemical substance of the proteinic enzymatic activity of a kind of deactivation, described protein is as for plant-growth or the translocator matter of surviving essential, biosynthetic enzyme, acceptor, signal transcription protein, structure gene product or translocator.In the context of the invention, inhibition is the chemical substance of the enzymatic activity of a kind of deactivation plant HMP-P kinases/TMP-PP enzyme.When being applied to plant, vegetable cell, plant seed or plant tissue, term used herein " weedicide " defines a kind of inhibition.

Isogenic: except can or not having hereditary identical plant the transgenosis difference because of existence.

Isolating: in the context of the invention, isolated DNA molecule or isolating enzyme are dna molecular or enzyme, exist owing to artificial its leaves self natural surroundings, and no longer are natural products therefore.A kind of isolated DNA molecule or enzyme can exist or be present in non-natural environment such as genetically modified host cell by purified form.

Mature protein: natural target is due to a kind of organoid such as chloroplast(id) and remove the protein of transit peptides at this place.

Minimal promoter: promoter element, especially TATA element, its non-activity or when the activation of no upstream, have the promoter activity that reduces greatly.When having suitable transcription factor, minimal promoter works and transcribes allowing.

The enzymic activity of modification: with the natural different enzymic activity (that is: naturally occurring enzymic activity when the direct or indirect mankind of nothing operate this activity) that exists in the plant, it tolerates the inhibition of the naturally occurring enzymic activity of inhibition.

Effectively connection is meant, if two sequences are so arranged the feasible expression that sequence can influence DNA sequences encoding of regulating, then regulates dna sequence dna and can be described as " being operably connected " or " associated " with coding RNA or protein DNA sequence.

Plant is meant any plant, especially spermatophyte.

Vegetable cell is meant structure and the physiology unit of plant, comprises protoplastis and cell walls.

Vegetable cell can be isolating unicellular or cultured cells form, or as the part of high orderly unit as for example plant tissue or plant organ.

Vegetable material is meant part, fruit, pollen, pollen tube, ovule, blastular, ovum, zygote, embryo, seed, cutting, cell or tissue cultivation or the arbitrary other parts or the plant product of leaf, stem, root, flower or flower.

Preceding albumen: natural target schedules the protein of organoid such as chloroplast(id), and still contains its transit peptides.

Recombinant DNA is meant a kind of combination that utilizes the dna sequence dna that recombinant DNA technology combines.

Recombinant DNA technology is meant and is used for method that dna sequence dna is linked together, as Sambrook etc., and 1989, Cold Spring Harbor is described in the NY:Cold Spring HarborLaboratory Press.

Significantly increase: a kind of increase that surpasses the enzymatic activity of measuring technology inherent limit of error, being preferably the above active enzymic activity of wild-type enzyme under inhibition exists of about twice or twice increases, more preferably from about more than 5 times or 5 times, more preferably from about more than 10 times or 10 times.

Be starkly lower than: the quantitative changeization that is meant enzymatic reaction product surpasses measuring technology inherent limit of error, is preferably below the wild-type enzyme active 1/2 or 1/2 of inhibition under not existing, more preferably from about below 1/5 or 1/5, more preferably from about below 1/10 or 1/10.

More broad sense is said, term " similar basically " is when being used in reference to nucleotide sequence, be meant a kind of nucleotide sequence corresponding to the reference nucleotide sequence, wherein corresponding sequence coding is a kind of has the polypeptide of substantially the same 26S Proteasome Structure and Function with the nucleotide sequence coded polypeptide of reference, for example, only there is the amino acid change that does not influence the polypeptide function.Expectation, similar basically is nucleotide sequence coded by the nucleotide sequence coded polypeptide of reference.Basically the expectation of homogeny per-cent between similar nucleotide sequence and the reference nucleotide sequence is at least 85%, more desirably at least 90%, preferably at least 92%, more preferably at least 95%, more preferably at least 97%, even more preferably at least 99%.Utilizing Skmith-Waterman series arrangement comparison algorithm to carry out sequence relatively (sees, as Waterman, M.S. computer biology introduction: collection of illustrative plates, sequence and genome (Introduction to Computational Biology:Maps, sequences andgenomes), Chapman ﹠amp; Hall, London:1995, ISBN 0-412-99391-0, or visit http://www-hto.usc.edu/software/seqaln/index.html).Use localS program (1.16 editions) with following parameter: coupling: 1, mispairing is penalized: 0.33, open the district and penalize: 2, extension area is penalized: 2.One can be under the following conditions and the reference nucleotide sequence hybridization to the nucleotide sequence of reference nucleotide sequence " similar basically ": 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, 1mM EDTA, uses 1X SSC, 0.1%SDS, 65 ℃ of washings by 50 ℃; More desirably, 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, 1mM EDTA, uses 0.5X SSC, 0.1%SDS, 65 ℃ of washings by 50 ℃; Even more desirably at 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, 1mM EDTA, uses 0.2X SSC, 0.1%SDS, 65 ℃ of washings by 50 ℃; Preferably at 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, 1mM EDTA, uses 0.1X SSC, 0.1%SDS, 65 ℃ of washings by 50 ℃.

Term " similar basically " when being used for protein herein, is meant the protein corresponding to reference protein matter, and wherein protein has the 26S Proteasome Structure and Function substantially the same with reference protein matter, as for example, only has the amino acid change that does not influence the polypeptide function.When being used for protein or aminoacid sequence, the expectation of homogeny per-cent between similar basically sequence and the reference protein matter is at least 65%, more desirably at least 75%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

Substrate: substrate is the natural identification of enzyme and be converted into the molecule of product in the bio-chemical pathway of natural its function of enforcement of a kind of enzyme therein, or the modified forms of this molecule, it also can be discerned by enzyme and be converted into product in the enzymatic reaction of similar naturally occurring reaction.

Synthetic is meant a kind of nucleotide sequence that is not present in the constitutional features in the native sequences that comprises.The quite similar artificial sequence of the normal codon distribution of for example a kind of G+C content and unifacial leaf and/or dicotyledonous gene is called as synthetic.

Tolerance: agent still continues the ability of normal growth or functionating when a kind of inhibition of contact or weeding.

Transform: a kind of method allogeneic dna sequence DNA transfered cell, tissue or plant.Should understand cell transformed, tissue or plant and not only contain the end product of method for transformation, also comprise its transgenosis filial generation.

Transgenosis: stably transform with recombinant DNA molecules, described molecule preferably comprises the suitable promotor that effectively is connected with target dna sequence.

An object of the present invention is to provide the method for identifying novel herbicide or improved weedicide.Another object of the present invention provides utilizes this new or improved weedicide to suppress the method for plant (as weeds) growth.Another aspect of the present invention provides improved crop plants, and it tolerates described new or improved weedicide.

The present invention discloses the correct nucleotide sequence of mustard genus (Arabidopsis) HMP-P kinases/TMP-PP enzyme gene first.Proteic nucleotides sequence is shown in SEQ ID NO:1 before the coding, and the nucleotides sequence of the mature protein that coding is inferred is shown in SEQ ID NO:3.The correct aminoacid sequence that mustard belongs to HMP-P kinases and the preceding albumen of TMP-PP enzyme is shown in SEQ ID NO:2, and the correct aminoacid sequence that the ripe mustard of inferring belongs to HMP-P kinases and TMP-PP enzyme is shown in SEQ ID NO:4.Therefore, the present invention comprises the nucleotide sequence that derives from plant, and its coding has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity.In a preferred embodiment, this class nucleotide sequence is from Arabidopis thaliana (Arabidopsis thaliana), in a further preferred embodiment, this class nucleotide sequence is identical or similar basically with the nucleotide sequence that is shown in SEQ ID NO:1 or SEQ ID NO:3, the enzyme that coding has HMP-P kinase activity or TMP-PP enzymic activity, its aminoacid sequence is identical with the aminoacid sequence that is shown in SEQ ID NO:2 or SEQ IDNO:4 or similar basically.

HMP-P kinases and TMP-PP enzyme are the enzymatic steps in the VitB1 biosynthetic pathway (as follows) from the beginning.From the beginning the VitB1 biosynthesizing finally causes the formation (being also referred to as VitB1 bisphosphate or VITMAIN B1) of thiaminpyrophosphate, the form of this cofactor sees (Schellenberger etc., 1997, Enzymology method (Meth.Enz.) in several enzymes, 279,131-146).This from the beginning biosynthetic pathway see in bacterium, yeast and the plant, but do not exist among the mankind.(Begley, T.P., 1996, national product report (Natl.Prod.Rep.), 13,177-186).The pyrophosphorylation of the single phosphoric acid of the kinase whose enzymatic activity catalysis of HMP-P 2-methyl-4-amino-5-oxymethylpyrimidine (HMP-P) forms 2-methyl-4-amino-5-oxymethylpyrimidine tetra-sodium (HMP-PP).In intestinal bacteria, this step is undertaken by the protein of thiD genes encoding.TMP-PP enzyme enzymatic activity corresponding to thiaminpyrophosphate from the beginning in the biosynthesizing thereafter to final step, the coupling of catalysis 4-methyl-5-(beta-hydroxyethyl) thiazole list phosphoric acid (THZ-P) and HMP-PP is to form VitB1 list phosphoric acid (TMP).In intestinal bacteria, this step is undertaken by the protein of thiE genes encoding.

Knowledge by the correct nucleotide sequence of HMP-P kinases/TMP-PP enzyme gene, the inventor shows, need provide external source VitB1 to have sudden change with the VitB1 auxotrophic mutation body that guarantees growth in the encoding sequence of its HMP-P kinases/TMP-PP enzyme gene.This has proved the importance of this gene without doubt at molecular level.By this class knowledge, may develop the screening method that screening can suppress the chemical of HMP-P kinase activity or TMP-PP enzymic activity.Because the importance of the enzyme of HMP-P kinases or TMP-PP enzyme genes encoding expects that also this class chemical suppresses growth or the existence of plant, is the potential fine herbicide therefore.Thereby an object of the present invention is to provide the method that the HMP-P kinases that utilizes purifying or TMP-PP enzyme are identified its inhibition, the vegetation growth of described inhibition useful as herbicides to suppress not expect, for example in the field of plant growth, especially important crop on the rural economy, such as corn and other cereals crop, as wheat, oat, rye, Chinese sorghum, rice, barley, broomcorn millet, sod grass and fodder grasses etc., and cotton, sugarcane, beet, oilseed rape and soybean etc.

The present invention includes a kind of mosaic gene, it comprises the effective promotor that is connected of nucleotide sequence of inventing the enzyme of HMP-P kinase activity or TMP-PP enzymic activity with code book.The present invention also comprises a kind of recombinant vectors, and it comprises mosaic gene according to the present invention, and wherein said carrier can be transformed into host cell with being stabilized.The present invention also comprises a kind of host cell, and it comprises support according to the present invention, and wherein said nucleotides sequence is listed in this cell and can expresses.Preferably a kind of according to host cell of the present invention, wherein said host cell is a kind of eukaryotic cell.More preferably according to host cell of the present invention, wherein said host cell is selected from insect cell, yeast cell and vegetable cell.It is further preferred that a kind of host cell, wherein said host cell is a kind of prokaryotic cell prokaryocyte.More preferably according to host cell of the present invention, wherein said host cell is a bacterial cell.

What also comprise is the biosynthetic plant protein of isolating participation VitB1, and wherein said protein has HMP-P kinase activity or TMP-PP enzymic activity.Preferably a kind of isolating protein, wherein said plant is an Arabidopis thaliana.More preferably according to protein of the present invention, wherein said protein has the HMP-P kinase activity.Especially preferred is a kind of according to protein of the present invention, and wherein said protein comprises and the identical or similar basically aminoacid sequence of aminoacid sequence shown in the SEQ ID NO:2.Especially preferred is a kind of protein, and wherein said protein comprises the aminoacid sequence shown in the SEQ ID NO:2.

Preferably according to protein of the present invention, wherein said protein has the TMP-PP enzymic activity.More preferably a kind of according to protein of the present invention, wherein said protein comprises and the identical or similar basically aminoacid sequence of aminoacid sequence shown in the SEQ IDNO:2.Especially preferred is a kind of protein, and wherein said protein comprises the aminoacid sequence shown in the SEQ ID NO:2.

In a preferred embodiment, the invention describes the method for the ability of a kind of inhibition plant-growth of identifying chemical to be measured or existence, comprise the steps: that (a) under its product synthetic condition of endonuclease capable catalysis, mixes the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture with HMP-P kinase substrate or TMP-PP enzyme substrates; (b) under the same conditions, chemical to be measured in second reaction mixture and enzyme are mixed with the substrate that is used for first reaction mixture, keep as the identical time in first reaction mixture; (c) be determined at the activity of enzyme in first and second reaction mixtures; And (d) when the activity of enzyme in second reaction mixture is lower than enzyme in (expectation is starkly lower than) first reaction mixture active, select the inhibition plant-growth of chemical to be measured or the ability of existence.In a preferred embodiment, the enzyme with HMP-P kinases or TMP-PP enzymic activity is preferably Arabidopis thaliana from plant.In a further preferred embodiment, enzyme with HMP-P kinases or TMP-PP enzymic activity is by identical or similar basically with the nucleotide sequence that is shown in SEQ ID NO:1 nucleotide sequence coded, or has and the identical or similar basically aminoacid sequence of aminoacid sequence that is shown in SEQ ID NO:2.In a further preferred embodiment, the kinase whose substrate of HMP-P is 2-methyl-4-amino-5-oxymethylpyrimidine phosphoric acid (HMP-P), and in a further preferred embodiment, the substrate of TMP-PP enzyme is 4-methyl-5-(beta-hydroxyethyl) thiazole phosphoric acid (THZ-P).In another preferred embodiment, the substrate of TMP-PP enzyme is 2-methyl-4-amino-5-oxymethylpyrimidine tetra-sodium (HMP-PP).In another preferred embodiment, the activity of enzyme is measured by measuring the TMP that produces in the reaction mixture.In another preferred embodiment, the ADP from ATP measures the activity of enzyme in the reaction mixture by measuring.The present invention has also described a kind of calibration method, comprise the steps: that (a) under its product synthetic condition of endonuclease capable catalysis, mixes the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture with HMP-P kinase substrate or TMP-PP enzyme substrates; (b) under the same conditions, chemical to be measured and enzyme-to-substrate in second reaction mixture are mixed, keep as the identical time in first reaction mixture; (c) be determined at the activity of enzyme in first and second reaction mixtures; Wherein, if the link coupled enzymic activity is starkly lower than enzymic activity in first reaction mixture in second reaction mixture, chemical can inhibitory enzyme activity.

The present invention has also described the method for the ability of a kind of inhibition plant-growth of identifying chemical to be measured and existence, comprise the steps: (a) under endonuclease capable catalysis TMP coupling synthetic condition, the enzyme that has the HMP kinase activity in first reaction mixture is mixed with HMP kinase substrate and TMP-PP enzyme substrates with the enzyme with HMP-P kinase activity or TMP-PP enzymic activity; (b) under the same conditions, chemical in second reaction mixture and enzyme-to-substrate are mixed, keep as the identical time in first reaction mixture; (c) be determined at the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first and second reaction mixtures; And (d) when the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in second reaction mixture is starkly lower than the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture active, select the inhibition plant-growth of chemical to be measured or the ability of existence.In a preferred embodiment, the enzyme with HMP-P kinases or TMP-PP enzymic activity is preferably Arabidopis thaliana from plant.In a further preferred embodiment, enzyme with HMP-P kinases or TMP-PP enzymic activity is by identical or similar basically with the nucleotide sequence that is shown in SEQ ID NO:1 nucleotide sequence coded, or has and the identical or similar basically aminoacid sequence of aminoacid sequence that is shown in SEQ ID NO:2.In a further preferred embodiment, the kinase whose substrate of HMP is HMP, and in a further preferred embodiment, the substrate of TMP-PP enzyme is THZ-P.In another preferred embodiment, the activity of enzyme is measured by measuring the TMP that produces in the reaction mixture.The present invention has also described a kind of calibration method, comprise the steps: (a) under endonuclease capable catalysis TMP coupling synthetic condition, the enzyme that has the HMP kinase activity in first reaction mixture is mixed with HMP kinase substrate and TMP-PP enzyme substrates with the enzyme with HMP-P kinase activity or TMP-PP enzymic activity; (b) under the same conditions, chemical and enzyme-to-substrate in second reaction mixture are mixed, keep as the identical time in first reaction mixture; (c) be determined at the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first and second reaction mixtures; Wherein, be starkly lower than the enzymic activity that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture if having the enzymic activity of HMP-P kinase activity or TMP-PP enzymic activity in second reaction mixture, chemical can suppress to have the enzymic activity of the enzyme of HMP-P kinase activity or TMP-PP enzymic activity.The present invention has also described the method for the ability of a kind of inhibition plant-growth of identifying chemical to be measured and existence, comprise the steps: (a) under endonuclease capable catalysis TMP coupling synthetic condition, the enzyme that has the THZ kinase activity in first reaction mixture is mixed with HMP-P kinase substrate and THZ kinase substrate with the enzyme with HMP-P kinase activity or TMP-PP enzymic activity; (b) under the same conditions, chemical in second reaction mixture and enzyme-to-substrate are mixed, keep as the identical time in first reaction mixture; (c) be determined at the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first and second reaction mixtures; And (d) when the enzymic activity that has HMP-P kinase activity or TMP-PP enzymic activity in second reaction mixture is starkly lower than the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture active, select the inhibition plant-growth of chemical to be measured or the ability of existence.In a preferred embodiment, the enzyme with HMP-P kinases or TMP-PP enzymic activity is preferably Arabidopis thaliana from plant.In a further preferred embodiment, enzyme with HMP-P kinases or TMP-PP enzymic activity is by identical or similar basically with the nucleotide sequence that is shown in SEQ ID NO:1 nucleotide sequence coded, or has and be shown in the identical or similar basically aminoacid sequence of SEQ IDNO:2 aminoacid sequence.In a further preferred embodiment, the kinase whose substrate of HMP-P is HMP-P, and in a further preferred embodiment, the kinase whose substrate of THZ is THZ.In another preferred embodiment, the activity with enzyme of HMP-P kinase activity or TMP-PP enzymic activity is measured by measuring the TMP that produces in the reaction mixture.The present invention has also described a kind of calibration method, comprise the steps: (a) under endonuclease capable catalysis TMP coupling synthetic condition, the enzyme that has the THZ kinase activity in first reaction mixture is mixed with THZ kinase substrate and HMP-P kinase substrate with the enzyme with HMP-P kinase activity or TMP-PP enzymic activity; (b) under the same conditions, chemical and enzyme-to-substrate in second reaction mixture are mixed, keep as the identical time in first reaction mixture; (c) be determined at the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first and second reaction mixtures; Wherein, be starkly lower than the enzymic activity that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture if having the enzymic activity of HMP-P kinase activity or TMP-PP enzymic activity in second reaction mixture, chemical can suppress to have the activity of the enzyme of HMP-P kinase activity or TMP-PP enzymic activity.

In a further preferred embodiment, the invention describes the method that a kind of evaluation has the chemical that suppresses HMP-P kinases in the plant or TMP-PP enzymic activity ability, comprise the steps: a) to obtain transgenic plant, plant tissue, plant seed or vegetable cell, preferred stable conversion, it comprises the non-natural nucleoside acid sequence that coding has the enzyme of HMP-P kinases or TMP-PP enzymic activity, and the enzymatic activity of energy overexpression HMP-P kinases or TMP-PP enzyme; B) with chemical application in transgenic plant, vegetable cell, tissue or its part, and isogenic non-conversion plant, vegetable cell, tissue or its part; C) growth or the survival behind mensuration transgenosis and non-conversion plant, vegetable cell, the organizations chemical; D) growth or the survival behind comparison transgenosis and non-conversion plant, vegetable cell, the organizations chemical.Desirably, chemical suppresses the growth or the survival of non-transgenic plant, vegetable cell, tissue or its part, and the growth or the survival of the isogenic transgenic plant of not obvious inhibition, vegetable cell, tissue or its part.In one embodiment, coding has the nucleotide sequence of enzyme of HMP-P kinases or TMP-PP enzymic activity from plant, is preferably Arabidopis thaliana, and is desirably identical or similar basically with the nucleotide sequence that is shown in SEQ IDNO:1 or SEQ ID NO:3.In a further preferred embodiment, the enzyme with HMP-P kinases or TMP-PP enzymic activity is shown in aminoacid sequence nucleotide sequence coded of SEQ ID NO:2 or SEQ ID NO:4 by coding.In another preferred embodiment, the enzyme with HMP-P kinases or TMP-PP enzymic activity has and the identical or similar basically aminoacid sequence of aminoacid sequence that is shown in SEQ ID NO:2 or SEQ ID NO:4.

The present invention also comprises the HMP-P kinases with modification or plant, plant tissue, plant seed and the vegetable cell of TMP-PP enzymic activity, its therefore tolerance under normal level, be the inhibition of the weedicide of inhibition to naturally occurring HMP-P kinase activity or TMP-PP enzymic activity.

The herbicide tolerant plant that the present invention is contained comprises otherwise may become those plants of the potential target of inhibition weedicide, important crop on the especially above-mentioned rural economy.According to the present embodiment, plant, plant tissue, plant seed or vegetable cell transform with a kind of recombinant DNA molecules, preferred stable conversion.Described recombinant DNA molecules comprises the suitable promotor that function is arranged that effectively is connected with nucleotide coding sequence in plant, HMP-P kinases or the TMP-PP enzyme modified of nucleotide coding sequence coding wherein can suppress the restraining effect of weedicide of the concentration of the HMP-P kinases of wild-type unmodified or TMP-PP enzymic activity under the enzyme tolerance normal circumstances of modification.HMP-P kinases of modifying or TMP-PP enzymic activity also can be given plant by the HMP-P kinases or the TMP-PP expression of enzymes that increase the wild-type herbicide sensitive, and the increase of expression is by the multiple copy that wild-type HMP-P kinases or TMP-PP enzyme gene are provided to plant or is higher than under the control of wild-type promotor overexpression wild-type HMP-P kinases in intensity or TMP-PP enzyme gene is realized.So transgenic plant, plant tissue, plant seed or the vegetable cell that produces can screen by conventional screening methods, separates thus, characterizes and cultivated the herbicide tolerant strain.Perhaps, also can use at random or rite-directed mutagenesis produces the herbicide tolerant strain.

Therefore, the invention provides with the dna molecular plant transformed, plant tissue, plant seed or the vegetable cell that contain from the isolating nucleotide sequence of plant, described nucleotide sequence coded enzyme with HMP-P kinase activity or TMP-PP enzymic activity, wherein this enzyme has HMP-P kinase activity or TMP-PP enzymic activity, wherein dna molecular is given plant, plant tissue, plant seed or vegetable cell and is tolerated a certain amount of weedicide, suppresses naturally occurring HMP-P kinase activity or TMP-PP enzymic activity under the amount normal circumstances of this weedicide.An embodiment according to the present embodiment, enzyme with HMP-P kinase activity or TMP-PP enzymic activity is by identical or similar basically with nucleotide sequence shown in SEQ ID NO:1 or the SEQ ID NO:3 nucleotide sequence coded, or has identical or similar basically with aminoacid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 aminoacid sequence.

The present invention also provides the method that suppresses plant-growth, comprises suppressing the chemical application of naturally occurring HMP-P kinase activity or TMP-PP enzymic activity in the step of plant in plant.In relevant therewith method, the present invention relates to a kind of crop or the field selectivity of plant method of suppressing weed growth at the crop seed that contains plantation, comprise the steps: (a) plantation herbicide tolerant crop or crop seed, it is plant or plant seed that tolerance suppresses the weedicide of naturally occurring HMP-P kinase activity or TMP-PP enzymic activity; And (b) use a certain amount of weedicide to the crop in field or crop seed and weeds, and institute's consumption suppresses naturally occurring HMP-P kinase activity or TMP-PP enzymic activity, and wherein, weedicide suppresses the growth of weeds, and the growth of not obvious inhibition crop.

By studying following invention description and non-limiting example, those skilled in the art can know others of the present invention and advantage.

From Arabidopis thaliana, identified obligate organotrophy defective type (organoauxotrophic) mutant that some need external source VitB1 or VitB1 precursor (that is: 4-methyl-5-(beta-hydroxyethyl) thiazole (THZ) and/or 2-methyl-4-amino-5-oxymethylpyrimidine (HMP)), and 4 locus positions (Li and Redei have been drawn, 1969, biochemical genetics (Biochemical Genetics), 3,163-170).Locus th1 mapping be positioned at karyomit(e) 1 (Koomneef and Hanhart, 1981, mustard belongs to Information services (Arab.Info.Service), 18,52-58).Although the definite position of VitB1 can not be determined by Koomneef and Hanhart in this district's group between pyrimidine and thiazole precursor and the th1 mutant, showed by biochemical method that the th1 mutant lacked TMP-PP enzyme enzymatic activity (Komeda etc. afterwards, 1988, plant physiology (Plant Physiol.), 88,248-250).

Recently, the BAC sequence in this zone of covering coloring body 1 (BAC F19G10) has been committed to GenBank.On this BAC, a gene has and the obviously similar sequence (nucleotide position 46133-48657 on the BAC sequence) of the aminoacid sequence of intestinal bacteria thiD and thiE.Based on this information, the contriver has separated mustard and has belonged to cDNA, its a kind of new protein of encoding: the aminoacid sequence in N-end structure territory (about Nucleotide 906 to the SEQ ID NO:1 with two functional structure territories, or the amino acid 302 of SEQ ID NO:2) has homology with HMP-P kinases (thiD), the aminoacid sequence of C-terminal structural domain (from about Nucleotide 925 of SEQ ID NO:1, or the amino acid 309 of SEQ ID NO:2) homology had with TMP-PP enzyme (thiE).Also in preceding 99 Nucleotide of SEQ ID NO:1, predicted a chloroplast transit peptides of inferring (preceding 33 amino acid of SEQ ID NO:2).

The contriver has also obtained the HMP-P kinases/TMP-PP enzyme sequence from two kinds of different th1 mutant, CS79 and CS3530 (mustard belongs to preservation center, Nottingham, Britain).They find all to exist sudden change in the HMP-P of two kinds of mutant kinases/TMP-PP enzyme gene.CS79 has the single nucleotide alteration by C to T at 188 places, position of SEQ ID NO:1, this amino acid with position 63 among the SEQID NO:2 becomes phenylalanine from Serine.CS3530 has 7 nucleotide deletions in SEQ ID NO:l position 259-265 or the position 260-266, and this amino acid with position 87 among the SEQ ID NO:2 becomes l-asparagine from Isoleucine.In this mutant, the codon heel of l-asparagine 87 is the TGA terminator codon of reading in the frame with 13 amino acid whose codons then.Sudden change should cause only having 100 amino acid whose proteinic translations, and described protein only contains sub-fraction and thiD homologous structural domain, lacks and thiE homologous structural domain.These results show that VitB1 auxotroph phenotype is because the sudden change in HMP-P kinases/TMP-PP enzyme gene shows that without doubt HMP-P kinases/TMP-PP enzyme gene is most important for plant, has probably represented the good target spot of weedicide.

Based on the cDNA sequence, the contriver further finds the incorrect montage joint of prediction in the BAC clone.The montage joint of first prediction causes having added at 383 places, SEQ ID NO:1 position 24 bases (Fig. 2) of intron sequences improperly, and the montage joint of second prediction causes the deletion of 24 bases between SEQ ID NO:1 position 1066 and 1089 and the interpolation of 9 wrong bases improperly.Infer based on this, HMP-P kinases/TMP-PP enzyme gene does not have operability (inoperative), can not be used for purposes of the present invention.

HMP-P kinases/TMP-PP enzyme gene also further is called the enzyme that coding has HMP-P kinase activity or TMP-PP enzymic activity.Preferred thus presentation code has the HMP-P kinases/TMP-PP enzyme gene of the single polypeptide of HMP-P kinase activity or TMP-PP enzymic activity.In a further preferred embodiment, the single polypeptide by HMP-P kinases/TMP-PP enzyme genes encoding has HMP-P kinase activity and TMP-PP enzymic activity.

The nucleotide sequence (shown in SEQ ID NO:3) of encoding mature HMP-P kinases/TMP-PP enzyme gene has been preserved in agricultural research institute preservation center (NRRL) of the international depositary institution that sets up according to the budapest treaty of the microbial preservation unit of the international recognition that is used for the patented procedure purpose on July 8th, 1998,1815 N, University Street, Peoria, Illinois 61604, the U.S., clone's title aththiDE-ctp, preserving number NRRL B-30040.

For the recombinant production of the kinases of HMP-P in host living beings/TMP-PP enzyme, the nucleotide sequence that coding is had the enzyme of HMP-P kinase activity or TMP-PP enzymic activity inserts a kind of expression cassette that is designed for selected host, and the importing reorganization produces among its host.Being fit to selected host's the particular adjustments sequence such as the selection of promotor, signal sequence, 5 ' and 3 ' non-translated sequence and enhanser belongs within those skilled in the art's the know-how.The molecule that gained contains each element that is connected with proper reading frame can be inserted in the carrier that can be transformed into host cell.Being used for suitable expression vector and method that protein reorganization produces is that this area is known, for the host, such as intestinal bacteria, yeast and insect cell (see as, Luckow and Summers, biology/technology (Bio/Technol.), 6:47 (1988)).Specific examples comprises plasmid, such as pBluescript (Stratagene, La Jolla, CA), pFLAG (InternationalBiotechnologies, Inc., New Haven, CT), pTrcHis (Invitrogen, La Jolla, CA) and rhabdovirus expression vector, for example those are genomic from Autographica californica nucleopolyhedrosis virus (AcMNPV).A kind of preferred baculovirus/insect system be the pVl1l392/Sf21 cell (Invitrogen, La Jolla, CA).

In a preferred embodiment, coding has the nucleotide sequence of enzyme of HMP-P kinase activity or TMP-PP enzymic activity from eukaryote, as yeast, but preferably from plant.In another preferred embodiment, nucleotide sequence is identical or similar basically with nucleotide sequence in being shown in SEQ ID NO:1 or SEQID NO:3, or the enzyme of encoding and having HMP-P kinase activity or TMP-PP enzymic activity, its aminoacid sequence is identical or similar basically with the aminoacid sequence that is shown in SEQ ID NO:2 or SEQ ID NO:4.The nucleotide sequence coded mustard that is shown in SEQ ID NO:l belongs to the preceding albumen of HMP-P kinases/TMP-PP enzyme, its aminoacid sequence is shown in SEQ ID NO:2, the coding mustard belongs to the nucleotide sequence that is shown in SEQ ID NO:3 of inferring ripe HMP-P kinases/TMP-PP enzyme, and its aminoacid sequence is shown in SEQ IDNO:4.In another preferred embodiment, nucleotide sequence is from prokaryotic organism, and preferred cell is as intestinal bacteria.In this case, have the enzyme of HMP-P kinase activity and the enzyme that has the TMP-PP enzymic activity or have a TMP-PP enzymic activity respectively by thiD and thiE genes encoding.

Utilize the multiple standards technical point from the HMP-P kinases/TMP-PP enzyme that produces with purification of Recombinant.Spendable concrete technology depends on the host living beings of use, and whether enzyme is designed to excretory, and this class factor of being familiar with of other those skilled in the art (see, as, Ausubel, F. etc. compile the 16th chapter, contemporary molecular biology method, John Wiley ﹠amp; Sons, Inc. (1994)).

HMP-P kinases/TMP-PP enzyme that reorganization produces can be used for multiple use.For example, it can be used for external calibration method, to screen the unidentified known weedicide chemical of its target position, is used for determining whether that it suppresses HMP-P kinase activity or TMP-PP enzymic activity.The external calibration method of this class also can be used as more generally screening, suppress the active chemical of this class enzymatic to identify, so it is the novel herbicide material standed for.In addition, the HMP-P of recombinant production kinases/TMP-PP enzyme can be used for illustrating the complex construction of these molecules, further characterizes the relation of itself and known inhibitor, with new inhibition weedicide of appropriate design and herbicide tolerant type enzyme.

A kind of external calibration method that is used to differentiate by the inhibition of the enzyme (as HMP-P kinases/TMP-PP enzyme) of essential plant gene coding, comprise step: a) when suspection exists for enzyme function inhibition, enzyme and its substrate reactions that will have HMP-P kinase activity or TMP-PP enzymic activity; B) do not exist the enzyme ' s reaction speeding when suspecting inhibition to compare under enzyme ' s reaction speeding in the time of will having the inhibition of suspecting and the similarity condition; And c) measures the enzymatic activity that the inhibition of whether suspecting suppresses kinase whose enzymatic activity of HMP-P or TMP-PP enzyme.The restraining effect of HMP-P kinase activity or TMP-PP enzymic activity reduced by TMP synthetic in the calibration method or suppress fully determine.In a preferred embodiment,, adopt the relatively amount of synthetic TMP in external calibration method of fluoroscopic examination, carry out this mensuration by under existence of candidate's inhibition and non-existent condition.In a further preferred embodiment, this mensuration is by under existence of candidate's inhibition and non-existent condition, the amount of the ADP that the comparison of employing absorbance detection forms in external calibration method.Preferred HMP-P kinase substrate is 2-methyl-4-amino-5-oxymethylpyrimidine phosphoric acid (HMP-P).Preferred TMP-PP enzyme substrates is 2-methyl-4-amino-5-oxymethylpyrimidine tetra-sodium (HMP-PP) and 4-methyl-5-(beta-hydroxyethyl) thiazole phosphoric acid (THZ-P).

By using the amount of link coupled HMP kinases/HMP-P kinases/HMP-P that TMP-PP enzyme calibration method increase HMP-P kinases can contact, increase the limit of detection of calibration method thus, the screening that is improved suppresses the method for the chemical of HMP-P kinase activity or TMP-PP enzymic activity.This class coupling calibration method comprises step: (a) under endonuclease capable catalysis TMP coupling synthetic condition, the enzyme that has the HMP kinase activity in first reaction mixture is mixed with HMP kinase substrate and TMP-PP enzyme substrates with the enzyme with HMP-P kinase activity or TMP-PP enzymic activity; (b) under the same conditions, chemical in second reaction mixture and enzyme-to-substrate are mixed, keep as the identical time in first reaction mixture; (c) be determined at the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first and second reaction mixtures; And (d) when the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in second reaction mixture is starkly lower than the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture active, select the inhibition plant-growth of chemical to be measured or the ability of existence.In a preferred embodiment, has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity from plant.In another preferred embodiment, enzyme with HMP-P kinase activity or TMP-PP enzymic activity is by identical or similar basically with the nucleotide sequence that is shown in SEQ ID NO:1 or SEQ ID NO:3 nucleotide sequence coded, or has and the identical or similar basically aminoacid sequence of aminoacid sequence that is shown in SEQ ID NO:2 or SEQ ID NO:4.In another preferred embodiment, the kinase whose substrate of HMP is HMP, and in another preferred embodiment, the substrate of TMP-PP enzyme is THZ-P.In another preferred embodiment, the activity of enzyme is measured by the TMP that produces in the assaying reaction mixture.

In addition, by using the amount of link coupled THZ kinases/HMP-P kinases/THZ-P that TMP-PP enzyme calibration method increase TMP-PP enzyme can contact, increase the limit of detection of calibration method thus, the screening that is improved suppresses the method for the chemical of HMP-P kinase activity or TMP-PP enzymic activity.This class coupling calibration method comprises step: (a) under endonuclease capable catalysis TMP coupling synthetic condition, the enzyme that has the THZ kinase activity in first reaction mixture is mixed with HMP-P kinase substrate and THZ kinase substrate with the enzyme with HMP-P kinase activity or TMP-PP enzymic activity; (b) under the same conditions, chemical in second reaction mixture and enzyme-to-substrate are mixed, keep as the identical time in first reaction mixture; (c) be determined at the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first and second reaction mixtures; And (d) when the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in second reaction mixture is starkly lower than the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture active, select the inhibition plant-growth of chemical to be measured or the ability of existence.In a preferred embodiment, has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity from plant.In another preferred embodiment, enzyme with HMP-P kinase activity or TMP-PP enzymic activity is by identical or similar basically with the nucleotide sequence that is shown in SEQ ID NO:1 or SEQ ID NO:3 nucleotide sequence coded, or has and the identical or similar basically aminoacid sequence of aminoacid sequence that is shown in SEQ ID NO:2 or SEQ ID NO:4.In another preferred embodiment, the kinase whose substrate of HMP-P is HMP-P, and in another preferred embodiment, the kinase whose substrate of THZ is THZ.In another preferred embodiment, the activity with enzyme of HMP-P kinase activity or TMP-PP enzymic activity is measured by the TMP that produces in the assaying reaction mixture.

In one embodiment, will be for example weedicide to be selected by the in-vitro screening evaluation be applied to plant with multiple concentration.Weedicide to be selected preferably sprays in plant.Weedicide to be selected writes down its effect to plant (for example death or growth-inhibiting) after using.

In another embodiment, a kind of calibration method of screening HMP-P kinase activity or TMP-PP enzymic activity inhibition is used transgenic plant, plant tissue, plant seed or vegetable cell, the nucleotide sequence that described material energy overexpression has HMP-P kinase activity or TMP-PP enzymic activity, wherein HMP-P kinases and TMP-PP enzyme have enzymatic activity in transgenic plant, plant tissue, plant seed or vegetable cell.Nucleotide sequence is preferably from eukaryote, such as yeast, but preferably from plant.In a preferred embodiment, nucleotide sequence is identical or similar basically with the nucleotide sequence that is shown in SEQ ID NO:1 or SEQ ID NO:3, or the enzyme of encoding and having HMP-P kinase activity or TMP-PP enzymic activity, its aminoacid sequence is identical or similar basically with the aminoacid sequence that is shown in SEQ ID NO:2 or SEQ ID NO:4.In another preferred embodiment, nucleotide sequence is from prokaryotic organism, and preferred bacterium is as intestinal bacteria.In this case, has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity respectively by thiD and thiE genes encoding.

Then with chemical application in transgenic plant, plant tissue, plant seed or vegetable cell, and be applied to isogenic non-conversion plant, plant tissue, plant seed or vegetable cell, measure the growth or the existence of transgenosis and non-conversion plant, plant tissue, plant seed or vegetable cell in applied chemistry product back, and relatively.

The invention still further relates to plant, plant tissue, plant seed and the vegetable cell of herbicide-tolerant, described weedicide suppresses naturally occurring HMP-P kinase activity or TMP-PP enzymic activity in these plants, and wherein tolerance is to give by the HMP-P kinase activity or the TMP-PP enzymic activity that change.HMP-P kinases/TMP-PP enzyme gene by extra wild-type herbicide sensitive is provided to plant, by in plant, expressing modified herbicide tolerant type HMP-P kinases/TMP-PP enzyme or the combination by these technology, increase wild-type herbicide sensitive type HMP-P kinases/TMP-PP enzyme, HMP-P kinase activity of Gai Bianing or TMP-PP enzymic activity can be given plant of the present invention thus.Representative plant comprises any plant that weedicide is used to plant with its normal purpose.Be preferably crop important on the rural economy, as cotton, soybean, oilseed rape, beet, corn, rice, wheat, barley, oat, rye, Chinese sorghum, broomcorn millet, sod grass and fodder grasses etc.

Increase HMP-P kinase activity or the TMP-PP enzymic activity that realizes change by expressing, cause the level of HMP-P kinases/TMP-PP enzyme in the vegetable cell at least enough to overcome the growth-inhibiting that causes by weedicide.The level of the enzyme of expressing generally is the twice of natural expression amount at least, preferably at least 5 times, and more preferably at least 10 times.Expressing increase can be owing to the multiple copy of wild-type HMP-P kinases/TMP-PP enzyme gene; Sudden change in the non-coding sequence of native gene, the adjusting sequence in the multiple appearance (that is: gene amplification) of encoding sequence or the vegetable cell in the gene.Plant with gene activity of this class change can obtain (see United States Patent (USP) 5162602 and United States Patent (USP) 4761373, be incorporated herein by reference) herein by this area direct screening of known plant.These plants also can obtain by the known gene engineering method in this area.By realizing also that with reorganization or chimeric dna molecule transformed plant cells herbicide sensitive type HMP-P kinases/TMP-PP enzyme expression of gene increases, wherein said dna molecular comprises the promotor that can drive dependency structure genetic expression in vegetable cell, and this promotor effectively is connected with the homology or the allos structure gene of coding HMP-P kinases/TMP-PP enzyme.Preferably, conversion is stable, and heritable transgenosis proterties is provided thus.The expression of B. modified herbicide tolerant type HMP-P kinases/TMP-PP enzyme

According to the present embodiment, in plant, there is the recombinant DNA molecules of suitable promotor function, that effectively be connected with encoding sequence stably to transform plant, plant tissue, plant seed or vegetable cell with containing, encode a kind of HMP-P kinases/TMP-PP enzyme of herbicide tolerant form of wherein said encoding sequence.The enzyme of herbicide tolerant form has at least one amino-acid substitution, interpolation or deletion, and it gives the tolerance to the weedicide of the enzyme of the not modified natural existence form of inhibition.Can screen transgenic plant, plant tissue, plant seed or the vegetable cell of generation like this by conventional triage techniques, separable thus, characterize and cultivate the herbicide tolerant strain.The method of the gene that obtains coding herbicide tolerant type HMP-P kinases/TMP-PP enzyme is described below.

A kind of general method comprises the direct or indirect mutafacient system to microorganism.For example, exercisable microorganism of a kind of heredity such as intestinal bacteria or yeast saccharomyces cerevisiae (S.cerevisiae) can be by with carrying out random mutagenesis in the body as UV light or methylsulfonic acid ethyl ester or methyl ester.Mutafacient system is Miller for example, molecular genetics experiment (Experiments in Molecular Genetics), ColdSpring Harbor Laboratory, Cold Spring Harbor, NY (1972); Davis etc., higher bacteria genetics (Advanced Bacterial Genetics), Cold Spring HarborLaboratory, Cold Spring Harbor, NY (1980); Sherman etc., yeast genetics method (Methods in Yeast Genetics), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1983); With United States Patent (USP) 4,975, described in 374.The microorganism that selection is used for mutagenesis contains normal inhibition responsive type HMP-P kinases/TMP-PP enzyme gene, and relies on and activity that this gene is given.There is the cell growth of sudden change down in the inhibition of the concentration that suppresses the unmodified gene.Screening mutant microbial bacterium colony (that is, show resistance to inhibition) better than the colony growth of mutant microbial not in the presence of inhibition is used for further analysis.Separate HMP-P kinases/TMP-PP enzyme gene by clone or pcr amplification, illustrate its sequence from these clones.The sequence of the gene product that coding changes is cloned back in the microorganism then, gives the ability of inhibition tolerance to confirm it.

The allelic method of sudden change herbicide tolerant that a kind of acquisition contains plant HMP-P kinases/TMP-PP enzyme gene comprises directly screening in the plant.For example, by will with the seed kind of the known method sterilization in this area on the flat board of the simple basic salt culture medium that contains the inhibition that concentration increases progressively, measuring the HMP-P kinases/TMP-PP enzyme gene pairs plant of sudden change such as the growth-inhibiting effect of mustard genus, soybean or corn.This concentration is in per 1,000,000 parts in 0.001,0.003,0.01,0.03,0.1,0.3,1,3,10,30,110,300,1000 and 3000 part the scope.The minimum concentration that repeatability detects obvious inhibition growth is used for experiment subsequently.Mensuration for those skilled in the art's minimum concentration is routine operation.

Utilize the mutagenesis of vegetable material to be increased in the frequency that occurs resistance allele in the screening colony.The seed material of sudden change is from multiple source, comprise chemistry or physical mutagenesis seed, or chemistry or physical mutagenesis pollen (Neuffer, the corn that is used for biological study, Sheridan compiles .Univ.Press, Grand Forks, ND, pp.61-64 (1982)), it is used for then to plant pollination, and collects the M1 mutant seed that obtains.Usually, belong to for mustard, by after the plant that obtains with chemical process (as ethyl methane sulfonate) or with the seed growth of physical method (gamma-rays or fast neutron) sudden change for seed-M2 seed (Lehle Seeds, Tucson, AZ), on the basic salt culture medium that contains the proper concn inhibition that is useful on screening tolerance type, plant plate with maximum 10000 seed/flat boards (10cm diameter).The seedling that continues growth and kept green in 7-21 days behind kind of plate is implanted into soil, grows to maturation and sets seeds.Test the tolerance of the filial generation of these seeds to weedicide.If the tolerance proterties is a dominance, its seed is separated into 3: 1/ resistances: the plant of responsive type is assumed to be at M2 for the resistance heterozygosis.The plant that produces whole resistance seeds is assumed to be at M2 and isozygotys for resistance.This sudden change to whole seed and the screening of its M2 filial generation seed also can be carried out other kind, for example soybean (seeing as United States Patent (USP) 5084082).Perhaps, wait that the sudden change seed that screens Herbicid resistant can obtain by using the pollen pollination through chemistry or physical means mutagenesis.

The hereditary basis of herbicide tolerant is that the confirmation of HMP-P kinases/this theory of TMP-PP enzyme gene of modifying is by further establishment as follows.At first.With based on SEQ ID NO:? shown in mustard belong to the primer of cDNA encoding sequence, or, utilize PCR to separate from the allelotrope of demonstration to HMP-P kinases/TMP-PP enzyme gene of the plant of inhibition resistance more preferably based on from the primer that does not change HMP-P kinases/TMP-PP enzyme gene order that is used for producing the allelic plant of tolerance.To equipotential genetic testing sequence with after determining there is sudden change in the encoding sequence, it gives the ability of plant to the inhibition resistance test allelotrope, transforms with the allelotrope of giving tolerance of inferring in this plant.This class plant can be mustard or it is grown to any other plant of HMP-P kinases/TMP-PP enzyme inhibitor sensitivity.The second, and the HMP-P kinases/TMP-PP enzyme gene mapping that draw to insert with respect to known limitation fragment length polymorphism (RFLP) (see, Chang etc. for example, institute of NAS reports 85:6856-6860 (1988); Nam etc., vegetable cell (Plant Cell) 1:699-705 (1989)).Utilize same tag to draw HMP-P kinases/TMP-PP enzyme inhibitor tolerance proterties collection of illustrative plates independently.When tolerance is because during sudden change in HMP-P kinases/TMP-PP enzyme gene, the tolerance proterties is plotted in the position with HMP-P kinases/TMP-PP enzyme gene location undistinguishable.

Kinases/the allelic method of TMP-PP enzyme gene herbicide tolerant type is by screening in plant cell cultures to another kind of acquisition HMP-P.With the plant tissue explant as, embryo, leaf dish etc., or the callus of active growth, or the suspension culture of target plant is grown in the substratum of the inhibition weedicide that exists concentration the to increase progressively inhibition analogue of laboratory environment (or be applicable to).In different cultures, record different extent of growth.In some culture, quick growth variant colony that grows even still lasting growth in the presence of the inhibition of normal inhibition concentration.With before inhibition contact tissue or the cell, handling with chemistry or physical method to increase grow the fast probability of variant appearance of this class.Separate and test the allelotrope of the HMP-P kinases of the giving tolerance/TMP-PP enzyme gene of inferring as described above.The allelotrope that then these is accredited as the conferring herbicide tolerance carries out genetic engineering modified, is beneficial to express and be transformed into plant.Perhaps, can be from contain these allelic tissues or cell culture aftergrowth.

Also have a kind of method to comprise, the plant HMP-P kinases of mutagenesis wild-type herbicide sensitive/TMP-PP enzyme gene in bacterium or yeast, culturing micro-organisms on the substratum that contains inhibition concentration inhibition is screened those bacterium colonies of growth in the presence of inhibition then subsequently.More specifically, with a kind of plant cDNA, the mustard of the HMP-P kinases/TMP-PP enzyme of for example encoding belongs to that cDNA is cloned into otherwise the microorganism that can lack selected gene activity.By several chemistry known in the art or Enzymology method microorganism transformed is carried out mutagenesis in vivo or vitro mutagenesis then, for example sodium bisulfite (Shortle etc., Enzymology method (Methods Enzymoi.) 100:457-468 (1983); Methoxyl group ammonia (Kadonaga etc., nucleic acids research (Nucleic AcidsRes.) 13:1733-1745 (1985); The directed saturation mutagenesis of oligonucleotide (Hutchinson etc., institute of NAS newspaper, 83:710-714 (1986); Or multiple polysaccharase mistake mix method (see, as, Shortle etc., institute of NAS newspaper, 79:1588-1592 (1982); Shiraishi etc., gene 64:313-319 (1988); With Leung etc., technology (Technique) 1:11-15 (1989).Select the bacterium colony of growing in the presence of the inhibition of normal inhibition concentration, in addition purifying repeats to rule.Its plasmid of purifying, and by it being transformed into again the microorganism that lacks HMP-P kinases/TMP-PP enzyme gene activity, the ability of inhibition tolerance is given in test.Determine thus to insert segmental dna sequence dna by the plasmid cDNA of this test.

Also can utilize the method that comprises vitro recombination (being also referred to as DNA reorganization) to obtain Herbicid resistant HMP-P kinases/TMP-PP enzyme.By DNA reorganization, will suddenly change (preferred random mutation) imports HMP-P kinases/TMP-PP enzyme gene.DNA reorganization also causes the reorganization and the rearrangement of sequence in HMP-P kinases/TMP-PP enzyme gene, the reorganization and the exchange of sequence between perhaps two or more different HMP-P kinases/TMP-PP enzyme gene.The feasible sudden change HMP-P kinases/TMP-PP enzyme gene that produces in 1,000,000 of these methods.Whether screening mutator gene or reorganization exist the character of expectation in the gene, as to the improved tolerance of weedicide, and give wide spectrum tolerance to different types of inhibition chemistry.Such screening is in those skilled in the art's routine operation scope.

A kind of reorganization dna molecular has been contained in the present invention, wherein said dna molecule encode has the HMP-P kinases/TMP-PP enzyme of enhanced herbicide tolerant, described weedicide suppresses HMP-P kinase activity or the TMP-PP enzymic activity by the template DNA molecule encoding, and described reorganization dna molecular template DNA molecule is thus derived.In addition, also contain a kind of mutant DNA molecule that obtains by reorganization template DNA molecule, described template DNA molecule encoding has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity, wherein, described mutant DNA molecule encoding has the HMP-P kinases/TMP-PP enzyme of enhanced herbicide tolerant, and this weedicide suppresses HMP-P kinase activity or the TMP-PP enzymic activity by described template DNA molecule encoding.

In a preferred embodiment, form sudden change HMP-P kinases/TMP-PP enzyme gene by at least a template HMP-P kinases/TMP-PP enzyme gene, wherein template HMP-P kinases/TMP-PP enzyme gene has been cut into the double-stranded random fragment of expectation size, and comprising the step that the double-stranded random fragment of gained colony is added one or more strands or double chain oligonucleotide, wherein said oligonucleotide comprises one and double-stranded random fragment same zone and one and the allogenic zone of double-stranded random fragment; Double-stranded random fragment of gained and oligonucleotide sex change are become single chain molecule; The incubation under such condition with gained single chain molecule colony and polysaccharase, this condition can cause described single chain molecule in this same area annealing, right to form the annealing fragment, described same area is enough to make the annealing fragment, and a right side causes duplicating of the opposing party, forms a kind of double-stranded polynucleotide of sudden change thus; Repeat the second and the 3rd step at least two circulations again, the gained mixture comprised that further circulation forms the double-stranded polynucleotide of another sudden change from the double-stranded polynucleotide of the sudden change in the third step that formerly circulates during wherein further round-robin second went on foot; Wherein, the double-stranded polynucleotide of sudden change are HMP-P kinases or the TMP-PP enzyme genes that has weedicide enhanced tolerance, and described weedicide suppresses naturally occurring HMP-P kinase activity or TMP-PP enzymic activity.

In another preferred embodiment, the template DNA molecule that has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity by at least two kinds of non-identical codings forms the dna molecular that the coding that suddenlys change has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity, comprises following steps: add at least a oligonucleotide that contains the zone identical with each template DNA molecule to the template DNA molecule; The sex change of gained mixture is become single chain molecule; The incubation under such condition with gained single chain molecule colony and polysaccharase, this condition can cause the annealing of oligonucleotide and template DNA molecule, and wherein being used for the condition that polysaccharase plays polymerization is the polymerization product that obtains corresponding to the part of template DNA molecule; Repeat the second and the 3rd step at least two circulations again, wherein the extension products that obtains in the 3rd step can cause the template DNA molecule be used for polymerization in next circulation; Form the double-stranded polynucleotide of sudden change thus, it comprises the sequence from the different templates dna molecular; Wherein, the double-stranded polynucleotide encoding of sudden change has the HMP-P kinases of weedicide enhanced tolerance or TMP-PP enzyme, and described weedicide suppresses HMP-P kinase activity or the TMP-PP enzymic activity by this template DNA molecule encoding.

In a preferred embodiment, in the concentration of double-stranded random fragment colony double center chain random fragment single kind 1% (weight) less than total DNA.In another preferred embodiment, the double-stranded polynucleotide of template comprise at least about 100 kinds of polynucleotide.In another preferred embodiment, the size of double-stranded random fragment is from about 5bp to 5kb.In another preferred embodiment, the 4th step of method comprises at least 10 circulations of step in repetition second and the 3rd step.This method is at for example Stemmer etc., (1994) nature, and 370:389-391, United States Patent (USP) 5,605,793 and Crameri etc., description is arranged among (1998) natural 391:288-291 and the WO 97/20078, be incorporated herein by reference herein.

In another preferred embodiment, the combination of any two or more different HMP-P kinases/TMP-PP enzyme gene is by Zhao for example etc., (1998) described in Nature Journal biotechnology fascicle (Nature Biotechnology) 16:258-261, by irregular extension method (StEP) vitro mutagenesis.Two or more HMP-P kinases/TMP-PP enzyme gene is as the template of pcr amplification, and the extension circulation of PCR reaction is preferably carried out under the temperature that is lower than the best polymerization temperature of polysaccharase.For example, when using optimum temps to be about 72 ℃ heat-stabilised poly synthase, the temperature expectation of extension is lower than 72 ℃, more preferably is lower than 65 ℃, preferably is lower than 60 ℃, and more preferably the temperature of extension is 55 ℃.In addition, the PCR reaction cycle time length of extension desirably is less than the used time of common this area, more desirably is less than 30 seconds, and preferably it is less than 15 seconds, and more preferably the time length of extension is 5 seconds.Only there is being the short dna fragment to be aggregated in the extension each time, making the template switching of extension products between the initiate dna molecule after each sex change and the anneal cycles, in extension products, producing multiple multi-form thus.The optimum cycle number of PCR reaction depends on HMP-P kinases/TMP-PP enzyme coding region length for the treatment of mutagenesis, and preferably more than 40 circulations, more preferably more than 60 circulations, the preferred use is higher than 80 circulations.As the best of using the known method in this area to measure each HMP-P kinases/TMP-PP enzyme assortment of genes is extended condition and best PCR number of cycles.Other PCR reaction parameter basically with commonly used identical in this area.The primer of amplified reaction preferably is designed to the dna sequence dna annealing outer with being positioned at HMP-P kinases/TMP-PP enzyme gene coded sequence, for example anneal with the dna sequence dna of the carrier that contains HMP-P kinases/TMP-PP enzyme gene, thus, the different HMP-P kinases/TMP-PP enzyme gene that is used for PCR reaction is preferably included in other carrier of branch.Primer desirably be positioned at the sequence annealing that is no more than 500bp with HMP-P kinases/TMP-PP enzyme encoding sequence distance, preferably be positioned at the sequence annealing that is no more than 200bp with HMP-P kinases/TMP-PP enzyme encoding sequence distance, more preferably be positioned at the sequence annealing that is no more than 120bp with HMP-P kinases/TMP-PP enzyme encoding sequence distance.Preferably, HMP-P kinases/TMP-PP enzyme encoding sequence is surrounded by restriction site, and described site is included in the dna sequence dna of amplification in the PCR reaction, can make things convenient for extension amplification outcome to go into appropriate carriers thus.

In another preferred embodiment, the HMP-P kinases/TMP-PP enzyme gene fragment with sticky end is as preparation as described in the WO98/05765.Sticky end is prepared by following method, by being connected with second kind of oligonucleotide corresponding to first kind of oligonucleotide of the part of HMP-P kinases/TMP-PP enzyme gene, described second kind of oligonucleotide is not present in this gene or corresponding to the part of following gene, described gene discord is adjacent corresponding to the gene of first kind of oligonucleotide, and wherein second kind of oligonucleotide contains at least a ribonucleotide.Utilize first kind of oligonucleotide as template, second kind of oligonucleotide prepares double-stranded DNA as primer.The excision ribonucleotide.The Nucleotide that is positioned at ribonucleotide 5 ' side also is removed, and causes double-stranded fragment to have sticky end.By ressembling this class fragment at random with being connected of the new gene order combination of gained.

Any HMP-P kinases/TMP-PP enzyme gene or any HMP-P kinases/TMP-PP enzyme assortment of genes can be used for vitro recombination of the present invention.For example, HMP-P kinases/TMP-PP enzyme gene is from plant, and as Arabidopis thaliana, for example HMP-P kinases/TMP-PP enzyme gene is as shown in SEQID NO:1 or SEQ ID NO:3.The gene of other suitable vitro recombination has from the HMP-P kinases of bacterium such as intestinal bacteria thiD or thiE gene/TMP-PP enzyme gene (Vander Horn etc., (1993) bacteriology magazine, 175,982-992; Backstrom etc., (1995) Journal of the American Chemical Society (J.Am.Chem.Soc.) 117,2351-2352), Salmonella typhimurium (Salmonella typhimurium) thiD gene (Petersen and Downs (1997) bacteriology magazine, 179,4894-4900), coarse arteries and veins spore mould (Neurospora crassa) thiD gene (Akiyama and Nakashima (1996), current genetics (Curr.Genet.) 30,62-67; Ouzonis and Kyrpides (1997) molecular evolution magazine (J.Mol.Evol.) 45,708-711) or yeast saccharomyces cerevisiae thiE gene (Nosaka etc., (1994) journal of biological chemistry (J.Biol.Chem.) 269 30510-30516), is hereby incorporated by.Use complete HMP-P kinases/TMP-PP enzyme gene or its part in the present invention.Sudden change HMP-P kinases/TMP-PP enzyme gene library as above-mentioned acquisition is cloned into suitable expression, and the gained carrier is transformed into suitable host, and algae for example is as the chlamydomonas class, in yeast or the bacterium.Suitably the host is preferably the host who lacks the HMP-P kinase activity originally, as coli strain NI500 (genetics storage center (Genetic Stock Center), New Haven, the U.S.), or the host of shortage TMP-PP enzymic activity, for example coli strain NI400 (Nakayama and Hayashi (1972) bacteriology magazine, 112:1118-1126).Cultivate on the substratum of the inhibition that contains inhibition concentration with the carrier transformed host cells that contains sudden change HMP-P kinases/TMP-PP enzyme gene library, be chosen in inhibition and have the bacterium colony of growth down.There is the bacterium colony of growth down in the inhibition of selecting inhibition concentration under normal circumstances, and in addition purifying repeats to rule.Its plasmid of purifying is measured the dna sequence dna that inserts fragment by the plasmid cDNA of this test.

A kind ofly identify that the calibration method of the modified HMP-P kinases/TMP-PP enzyme gene of tolerance inhibition can be as identifying HMP-P kinase activity or identical calibration method (the inhibition calibration method of TMP-PP enzymic activity inhibition, on seeing) carry out, except carrying out following modification: the first, the HMP-P kinases/TMP-PP enzyme of sudden change substitutes the wild-type HMP-P kinases/TMP-PP enzyme in the inhibition calibration method in a reaction mixture.The second, all there is the inhibition of wild-type enzyme in each reaction mixture.The 3rd, relatively sudden change active (activity that has inhibition and mutant enzyme) and sudden change active (activity that has inhibition and wild-type enzyme) are measured when comparing with the activity of not suddenling change, and whether observe that enzymic activity significantly increases in the sudden change activity.The sudden change activity is the measurement activity of any mutant enzyme when having suitable substrate and inhibition.The sudden change activity is not the measurement activity of any wild-type enzyme when having suitable substrate and inhibition.Significantly increase and be defined as a kind of increase that surpasses the enzymatic activity of measuring technology inherent limit of error, it is above in active active the increasing of the wild-type enzyme in the presence of inhibition to be preferably about twice or twice, more preferably from about more than 5 times or 5 times, the more preferably from about increase more than 10 times or 10 times.

Except being used to produce herbicide tolerant type plant, the gene of coding herbicide tolerant HMP-P kinases/TMP-PP enzyme also can be used as the selective marker in the vegetable cell method for transformation.For example, with transgenosis plant transformed, plant tissue, plant seed or vegetable cell also available code are a kind of can be by the gene transformation of HMP-P kinases/TMP-PP enzyme expression of plants, that change.Cell transformed is moved in the substratum, wherein contains the enzyme inhibitor of the amount of the vegetable cell existence that is enough to suppress not express modified enzyme, at this, only has cell transformed to survive.This method is applicable to any vegetable cell that any energy transforms with modified HMP-P kinases/TMP-PP enzyme coding gene, and can use with any target transgenosis.The expression of the gene of transgenosis and modification can or divide other promoters driven by the identical promoters that function is arranged in vegetable cell.

Preferably form the method for the HMP-P kinases of the present invention/TMP-PP enzyme gene of sudden change.

Further preferably form the method for described sudden change HMP-P kinases/TMP-PP enzyme gene.

More preferably form the method for the dna molecular of the present invention's sudden change that suddenlys change, one of them template DNA molecule is from eukaryote.Especially preferred is method of the present invention, and wherein said eukaryote is a plant.

Method more preferably of the present invention, the kind of wherein said template DNA molecule is identical or substantially similar with the nucleotide sequence shown in the SEQID NO:1.

More preferably the coding that is obtained by the inventive method has the mutant DNA molecule of the enzyme of HMP-P kinase activity or TMP-PP enzymic activity, wherein said mutant DNA molecule encoding has the HMP-P kinases/TMP-PP enzyme of enhanced herbicide tolerant, and described weedicide suppresses HMP-P kinase activity or the TMP-PP enzymic activity by described template DNA molecule encoding.

Utilize conventional recombinant DNA technology, a kind of wild-type or herbicide tolerant type HMP-P kinases/TMP-PP enzyme gene integration can be gone in plant or the bacterial cell.Usually, this comprise utilize this area standard cloning process will encode the dna molecular of HMP-P kinases/TMP-PP enzyme insert a kind of for this dna molecular the expression system for allos (being improper existence).Carrier contains for the protein coding sequence that inserts transcribes and translates essential element in containing the host cell of described carrier.Can use a large amount of carrier systems known in the art, for example the virus of plasmid, phage virus and other modification.The component of expression system also can be modified to increase and be expressed.For example, can utilize sequence, nucleotide subsitution or other modification of brachymemma.Can utilize expression system known in the art to transform the vegetable cell of any crop under proper condition.The transgenosis that contains wild-type or herbicide tolerant type HMP-P kinases/TMP-PP enzyme gene preferably is stabilized the genome that transforms and be integrated into host cell.In a further preferred embodiment, the transgenosis that contains wild-type or herbicide tolerant type HMP-P kinases/TMP-PP enzyme gene is positioned on the carrier of self-replicating.The carrier example of self-replicating is a virus, especially twin (gemini) virus.Transformant is renewable to be complete plant, makes the selected form of HMP-P kinases/TMP-PP enzyme gene give the transgenic plant herbicide tolerant.A. make up the requirement of expression of plants box

At first in expression cassette, after the suitable promotor that can in plant, express, assemble the gene order that preparation is expressed in transgenic plant.These expression cassettes also can comprise other any sequences that transgene expression is required or select.These sequences include but not limited to, for example, transcription terminator, strengthen the outside sequence of expressing, as intron, close key sequence and be used for gene product is directed to the sequence of specific cells device and cellular compartment.These expression cassettes easily can be transferred in the above-mentioned plant conversion carrier then.Be the description of the heterogeneity of typical expression cassette below.1. promotor

The selection of the promotor of using in expression cassette will determine genetically modified room and time expression pattern in the transgenic plant.The promotor of selecting will specific cellular type (as leaf epidermal cell, mesophyll cell, root chrotoplast) or in particular organization or organ (as root, leaf or flower) express transgenic, this selection will reflect the position of the hope that gene product accumulates.Perhaps, selected promotor can be expressed by guiding gene under multiple inductive condition.Difference aspect the ability that promotor promptly promotes to transcribe in its intensity.According to the host cell systems of using, can use in the multiple suitable promotor known in the art any.For example, CaMV 35S promoter, rice actin promoter or ubiquitin promotor can be used for constitutive expression.For example, the chemical induction type PR-1 promotor that derives from tobacco or Arabidopis thaliana can be used for adjustment type and expresses (see, for example, U.S. Patent number 5,689,044).2. transcription terminator

Multiple transcription terminator all can use in expression cassette.They are responsible for exceeding Transcription Termination outside the transgenosis and correct polyadenylation thereof.Suitable transcription terminator is the known transcription terminator that works in plant, comprises CaMV 35S terminator, tml terminator, nopaline synthase terminator and pea rbcS E9 terminator.They can be used in unifacial leaf and the dicotyledons.3. strengthen or regulate the sequence of expression

Found that many sequences can strengthen the genetic expression in the transcription unit, these sequences can be used in combination with gene of the present invention and strengthen its expression in transgenic plant.For example, the intron of multiple intron sequences such as corn Adh1 gene shows to strengthen expression, especially in monocot plant cell.In addition, knownly multiplely also can strengthen expression, and it is effective especially in the dicotyledons cell by viral deutero-untranslated leader.4. encoding sequence optimization

Be used for encoding sequence by change, encoding sequence that can the selected gene of genetic modification at purpose crop species optimum expression.Modify encoding sequence be implemented in specific do in the species method of optimum expression be well-known (see, for example, people such as Perlak, newspaper (Proc.Natl.Acad.Sci.USA) 88:3324 (1991) of institute of NAS; People such as Koziel, biotechnology (Bio/technol.) 11:194 (1993)).

5. the guiding of gene product in the cell

Known multiple in the plant be used to the to lead mechanism of gene product that is present in has more or less characterized the sequence of controlling these mechanism performance functions.For example, by seeing the aminoterminal signal sequence control of multiple proteins, it is cut in the chloroplast(id) input process gene product to the guiding of chloroplast(id), with produce mutein (as Comai etc., journal of biological chemistry, 263:15104-15109) (1988).Other gene product is positioned other organoid, as plastosome and peroxysome such as Unger etc., and molecular biology of plants, 13:411-418 (1989).The cDNA of these products of encoding also can be handled, to influence the guiding of heterologous gene products to these organoids.In addition, characterized and caused the sequence of gene product to the guiding of other cell element.The N-terminal sequence is responsible for the orientation to ER, apoplast, and from aleurone cell's exocytosis (Kohlar and Co) vegetable cell 2:769-783 (1990).In addition, N-terminal sequence and C-terminal sequence are responsible for the vacuole product (Shinshi etc., molecular biology of plants, 14:357-368, (1990)) of gene product.By the fusion of above-mentioned suitable targeting sequencing and target transgenic sequence, any organoid or cell unit may lead transgene product.B. the structure of plant conversion carrier

The multiple conversion carrier that can be used for Plant Transformation is that the technician in Plant Transformation field is known, and the gene relevant with the present invention can be used in combination with any of these carrier.Preferred transformation technology and the target species that is used to transform are depended in the selection of carrier.For some target species, preferred different microbiotic or weedicide selective markers.The conventional selective marker of using comprises npt II gene (Messing and Vierra, gene (Gene) 19:259-268 (1982) that gives kantlex and associated antibiotic resistance in the conversion; People such as Benvan, nature (Nature) 304:184-187 (1983)), the bar gene of conferring herbicide phosphinothricin resistance (people such as White, nucleic acids research 18:1062 (1990), people such as Spencer, theoretical and applied genetics (Theor.Appl.Genet.) 79:625-631 (1990)), give the antibiotic hygromycin resistance hph gene (Blochinger and Diggelmann, molecular cytobiology 4:2929-2931), allow manA gene (Miles and Guest (1984) the gene 32:41-48 that in the presence of seminose, just selecting; 1099-1104 (1983)) and the EPSPS gene of conferring glyphosate resistance (U.S. Patent number 4,940,935 and 5,188,642) U.S. Patent number 5,767,378) (people such as Bourouis, EMBO is (7) J.2:, to give the dhfr gene of methotrexate resistance.1. be applicable to the carrier that Agrobacterium (Agrobacterium) transforms

Many carriers can be used for using the conversion of agrobacterium tumefaciens (Agrobacterium tumefaciens).They generally have at least one T-DNA border sequence, and comprise carrier and pXYZ as pBIN19 (Bevan, nucleic acids research (1984)).The typical carriers that is applicable to Agrobacterium-mediated Transformation comprises binary vector pCIB200 and pCIB2001, and binary vector pCIB10 and Totomycin selection derivative thereof.(see, for example, U.S. Patent number 5,639,949).2. be applicable to the carrier of non-Agrobacterium-mediated Transformation

Do not use the conversion of agrobacterium tumefaciens to avoid needs, so except that the above-mentioned carrier that comprises the T-DNA sequence, can use the carrier that lacks these sequences to T-DNA sequence in the selected conversion carrier yet.The transformation technology that does not rely on Agrobacterium comprises the conversion of absorbing (as PEG and electroporation) and microinjection by particle bombardment, protoplastis.The selection of carrier depends primarily on the preferred of kind to be transformed.The typical carriers that is applicable to non-Agrobacterium-mediated Transformation comprises pCIB3064, pSOG19 and pSOG35 (see, for example, U.S. Patent number 5,639,949).C. transformation technology

The purpose encoding sequence promptly is transformed in the vegetable cell after being cloned in the expression system.Plant Transformation and regenerated method are well known in the art.For example, the Ti-plasmids carrier has been used for sending of foreign DNA to be passed, and directly DNA picked-up, liposome, electroporation, microinjection and microparticle bombardment.In addition, the also bacterium transformed plant cells of available Agrobacterium.

The transformation technology that is used for dicotyledons is well known in the art, and comprises based on the technology of Agrobacterium and does not need the technology of Agrobacterium.The technology of non-Agrobacterium comprises the direct picked-up to exogenous genetic material of protoplastis or cell.This can be by the mediation of PEG or electroporation sending of picked-up, particle bombardment mediation pass or microinjection is finished.In all situations, all use standard technique well known in the art to make cell transformed be regenerated as whole plants.

The conversion of most of monocotyledons kinds has now also become routine.Preferred technology comprises uses PEG or electroporation technology direct metastatic gene in protoplastis, to the particle bombardment of callus, and agriculture bacillus mediated conversion.

Wild-type of the present invention or the HMP-P kinases/TMP-PP enzyme gene that changes form can be used to the multiple variant conferring herbicide tolerance to vegetable cell, comprise those gymnosperms, monocotyledons and dicotyledons.Although gene can be inserted into any vegetable cell that falls into the scope of the invention, especially at the crop plants cell, as rice, wheat, barley, rye, corn, Ma Lingzhu, Radix Dauci Sativae, sweet potato, beet, pea, beans, witloof, lettuce, Caulis et Folium Brassicae capitatae, cauliflower, asparagus broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, Radix Dauci Sativae, summer squash, pumpkin, pumpkin, cucumber, apple, pears Quinces Quince, muskmelon, plum, cherry, peach, honey peach, apricot, strawberry, grape, raspberry, blackberry, blueberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, Chinese sorghum and sugarcane.

The high level expression of wild-type HMP-P kinases/TMP-PP enzyme gene and/or herbicide tolerant type HMP-P kinases/TMP-PP enzyme expression of gene are given the herbicide tolerant of plant, and other all can be incorporated in the plant lines by this area known breeding method and technology the feature of production and quality-critical.

When herbicide tolerant HMP-P kinases/TMP-PP enzyme allele gene obtains by the direct screening in crop plants or plant cell cultures, wherein by the renewable crop plants of described plant cell cultures, can utilize traditional breeding technology that it is moved in the commercial variant, with cultivation herbicide tolerant crop, and need not genetically engineered allelotrope and it is transformed into plant.Sequence summary SEQ ID NO:1 in the sequence table, mustard belongs to the preceding proteic dna sequence dna SEQ ID NO:2 of HMP-P kinases/IMP-PP enzyme, mustard belongs to the preceding proteic aminoacid sequence SEQ ID NO:3 of HMP-P kinases/TMP-PP enzyme, the ripe mustard of inferring belongs to the dna sequence dna SEQ ID NO:4 of HMP-P kinases/TMP-PP enzyme, the ripe mustard of inferring belongs to the aminoacid sequence SEQ ID NO:5 of HMP-P kinases/TMP-PP enzyme, oligonucleotide aththiDEforSEQ ID NO:6, oligonucleotide aththiDErevSEQ ID NO:7, preserving number preservation date aththiDE-ctp NRRL B-30040 on July 8th, 1998 is cloned in oligonucleotide aththiDEfor-ctp preservation

Further describe the present invention with reference to following detailed embodiment.The purpose that these embodiment are provided only is explanation, is not intended to restriction except as otherwise noted.

Embodiment

Standard DNA used herein and molecule clone technology are that this area is known, see as Sambrook etc., molecular cloning, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY (1989), and T.J.Silhavy, M.L.Berman and L.W.Enquist, gene fusion experiment (Experiments with Gene Fusions), Cold Spring HarborLaboratory, Cold Spring Harbor, NY (1984), and Ausubel, F.M. etc., molecular biology modernism (Current Protocols in Molecular Biology) GreenePublishing Assoc.and Wiley-Interscience publishes described in (1987).The embodiment 1 recombinant plant HMP-P kinases/expression of TMP-PP enzyme in intestinal bacteria

The acquisition and the environmental cDNA of a kind of Arabidopis thaliana Landsberg library of having increased (Minet etc., (1992) plant magazine (Plant J.) 2,417-422).By SEQ ID NO:? designed the PCR primer of the Arabidopis thaliana HMP-P kinases/TMP-PP zymoprotein encoding sequence that is used to increase, in order to from plasmid library, to utilize Pfu archaeal dna polymerase (Stratagene) amplification HMP-P kinases/TMP-PP enzyme encoding sequence.The structure that comprises the preceding proteic coding region of HMP-P kinases/TMP-PP enzyme in order to increase, use primer aththiDEfor (5 ' ggt att gag ggt cgcatg aat agc tta gga gga at 3 ', SEQ ID NO:5) and aththiDErev (5 ' aga ggagag tta gag cct caa att ccc ctt ttg ctc tct tta a 3 ', SEQ ID NO:6).To infer the structure of ripe HMP-P kinases/TMP-PP enzyme in order increasing, to use primer aththiDErev and aththiDEfor-ctp (5 ' ggt att gag ggt cgc tta aca gtg gcg gga tca gat 3 ', SEQ ID NO:7).Before albumen and the coding region of inferring mature protein gone into expression vector pET35b (+) Xa/LIC (Novagen) by subclone, and adopt the method for manufacturers all to be transformed into e. coli bl21 (DE3) (Novagen) by chemical conversion.The activity calibrating of embodiment 2 HMP-P kinases and TMP-PP enzyme is analyzed

The calibrating of the activity of HMP-P kinases and TMP-PP enzyme is analyzed and is come up from Komeda substantially etc., (1988) plant physiology (Plant Physiol.) 88,248-250.Reaction volume is preferably as described below, but can need to change according to experiment.0.01-1.0 * 10 ^-3The enzyme with HMP-P kinase activity or TMP-PP enzymic activity of unit (activity unit is defined as the amount that produces the required enzyme of 1mmol/ minute product) is with 10 ^-3-10 ^-5M (preferably 10 ^-4M) 2-methyl-4-amino-5-oxymethylpyrimidine phosphoric acid (HMP-P) and 10 ^-3-10 ^-5M (preferably 10 ^-4M) 4-methyl-5 (beta-hydroxyethyl) thiazole phosphoric acid (THZ-P) is mixed in 50 mMTris-HCl (pH 7.0-8.0, preferred 7.4) of final volume 100 mL, 1-20mM (preferred 10 mM) MgCl ₂, and among 0.1-10mM (preferred 1mM) ATP.The single phosphoric acid of VitB1 (TMP) is preferably by Kawasaki etc., (1990) bacteriology magazine, and 172, the method for 6145-6147 is measured, and adds 10% (w/v) metaphosphoric acid or 20% (w/v) trichoroacetic acid(TCA) (pH 1.4) of 50 mL.Centrifugal mixture is removed sedimentary protein then.Shift out 100ml supernatant liquor equal portions, add the 0.3M cyanogen bromide of 50ml, mix, add the 1M NaOH of 100ml subsequently.Measure fluorescence intensity of solution with excitation wavelength 360 ± 10nm and emission wavelength 430 ± 10nm.

Perhaps, the formation by the quantitative ADP of linked reaction method.In this case, add 3.5 unit pyruvate kinases, the serum lactic dehydrogenase of 4.7 units, 1.0mM phosphoenolpyruvic acid and 0.2mM NADH measure absorbancy in 340nm.Embodiment 3 coupling HMP kinases and HMP-P kinases and the calibrating of TMP-PP enzymic activity are analyzed the calibrating of HMP kinases and are analyzed

Follow the tracks of the conversion of HMP by detecting the ADP that forms simultaneously to HMP-P.Utilize pyruvate kinase and serum lactic dehydrogenase (reagent enzyme), detect in the presence of phosphoenolpyruvic acid (PEP) NADH to NAD ⁺Conversion, follow the tracks of the formation of ADP.Monitor at 340nm.Pyruvate kinase and PEP promote the regeneration of ATP by ADP.ATP is the substrate that HMP kinases and HMP-P kinases all need.The calibrating analysis buffer is for having added 10 mM MgCl ₂50 mM Tris-HCl, pH 7.5 (buffer A).HMP-P kinases and TMP-PP enzyme

Analyze HMP-P kinases and TMP-PP enzyme for calibrating, substrate HMP-P and THZ-P need be provided.HMP-P is produced to the conversion of HMP-P by HMP in the same reaction mixture.If add NADH, utilize the calibrating of HMP kinases to analyze and to follow the tracks of this conversion.When transforming to HMP-P, HMP carries out (about 50mM) when abundant, adding HMP-P kinases, TMP-PP enzyme and THZ-P.Produce the back at HMP-P and add HMP-P kinases, TMP-PP enzyme and THZ-P, guarantee initial HMP-P constant concentration in all reacting holes.By Kawasaki etc., (1990) bacteriology magazine, 172, the method for 6145-6147 is identified the amount of the TMP that forms.After TMP produces time enough (general 15 minutes), stop enzyme reaction with metaphosphoric acid or TCA.Oxidation TMP under alkaline condition.Measure the fluorescence of gained thiachrome derivative with excitation wavelength 360 ± 10nm and emission wavelength 430 ± 10nm.Calibration method

No matter analysis is all examined and determine with same procedure in the source of enzyme.In the 96 hole droplet plates of 300ml, examine and determine analysis.Total calibrating analytical reaction volume is 100ml.Make that with the mixed substrate final concentration is (in the droplet plate): HMP (100 mM), ATP (1000 mM), PEP (1000 mM), and NADH (200 mM).The 10X substrate mixture can move in the 10ml/ hole.Also but mix reagent enzyme and HMP kinases are to add simultaneously.The recommended amounts of the ADP detection/reconstituted mixt of each reaction is 1.0 unit pyruvate kinases and 1.0 unit serum lactic dehydrogenases.This can be used as guidance, and according to experience adjustments enzyme amount.After the HMP kinase reaction is finished with about 5mM/ minute speed responsing (within 10-15 minute), add THZ-P to final concentration 50mM, and add HMP-P kinases and TMP-PP enzyme.(by the activity decision of HMP-P kinases and TMP-PP enzyme) adds 50 mL 10% (w/v) metaphosphoric acids or 20% (w/v) trichoroacetic acid(TCA) (pH 1.4) termination reaction behind the certain intervals.Centrifugal culture plate, precipitating proteins moves into other droplet plate with supernatant liquor.The 0.3M cyanogen bromide that adds 50ml mixes, and adds the 1M NaOH of 100ml subsequently.Read the orifice plate value with excitation wavelength 360 ± 10nm and emission wavelength 430 ± 10nm by fluorophotometric droplet plate reader.Use single thiamine phosphoric acid to be standard.

Single thiamine phosphoric acid, ATP, PEP, NADH, metaphosphoric acid, trichoroacetic acid(TCA), cyanogen bromide and NaOH can derive from Sigma Chemicals.Utilize Schellenberger etc., (Hoppe-Seyler ' Z.Physiol.Chem. (1967) 348, method 501-505) is synthesized HMP, according to Leder etc., (1970) Enzymology method, 18A, the method for 166-167 is synthesized THZ-P.Embodiment 4 link coupled THZ kinases and HMP-P kinases and the calibrating of TMP-PP enzymic activity are analyzed the calibrating of A.THZ kinases and are analyzed

Follow the tracks of the conversion of THZ by detecting the ADP that forms simultaneously to THZ-P.Utilize pyruvate kinase and serum lactic dehydrogenase (reagent enzyme), detect in the presence of phosphoenolpyruvic acid (PEP) NADH to NAD ⁺Conversion, follow the tracks of the formation of ADP.Monitor at 340nm.Pyruvate kinase and PEP promote the regeneration of ATP by ADP.ATP is the substrate that THZ kinases and HMP-P kinases all need.The calibrating analysis buffer is for having added 10 mM MgCl ₂Buffer A.B.HMP-P kinases and TMP-PP enzyme

Analyze HMP-P kinases and TMP-PP enzyme for calibrating, substrate HMP-P and THZ-P need be provided.THZ-P is produced to the conversion of THZ-P by THZ in the same reaction mixture.If add NADH, utilize the calibrating of THZ kinases to analyze and to follow the tracks of this conversion.When transforming to THZ-P, THZ carries out (about 20mM) when abundant, adding HMP-P kinases, TMP-PP enzyme and HMP-P.Produce the back at THZ-P and add HMP-P kinases, TMP-PP enzyme and HMP-P, guarantee initial THZ-P constant concentration in all reacting holes.By Kawasaki etc., (1990) bacteriology magazine, 172, the method for 6145-6147 is identified the amount of the TMP that forms.After TMP produces time enough (general 15 minutes), stop enzyme reaction with metaphosphoric acid or TCA.Oxidation TMP under alkaline condition.Measure the fluorescence of gained thiachrome derivative with excitation wavelength 360 ± 10nm and emission wavelength 430 ± 10nm.C. calibration method

No matter analysis is all examined and determine with same procedure in the source of enzyme.In the 96 hole droplet plates of 300ml, examine and determine analysis.Total calibrating analytical reaction volume is 100ml.Mix substrate to scale and make that final concentration is (in the droplet plate): THZ (50 mM), ATP (5mM), PEP (1000mM), and NADH (200 mM).The 10X substrate mixture can move in the 10ml/ hole.Also but mix reagent enzyme and HMP kinases are to add simultaneously.The recommended amounts of the ADP detection/reconstituted mixt of each reaction is 1.0 unit pyruvate kinases and 1.0 unit serum lactic dehydrogenases.This can be used as guidance, and according to experience adjustments enzyme amount.After the THZ kinase reaction is finished with about 5mM/ minute speed responsing (within 5-10 minute), add HMP-P to final concentration 100mM, and add HMP-P kinases and TMP-PP enzyme.(by the activity decision of HMP-P kinases and TMP-PP enzyme) adds 50 mL 10% (w/v) metaphosphoric acids or 20% (w/v) trichoroacetic acid(TCA) (pH 1.4) termination reaction behind the certain intervals.Centrifugal culture plate, precipitating proteins moves into other droplet plate with supernatant liquor.The 0.3M cyanogen bromide that adds 50ml mixes, and adds the 1M NaOH of 100ml subsequently.Read the orifice plate value with excitation wavelength 360 ± 10nm and emission wavelength 430 ± 10nm by fluorophotometric droplet plate reader.Use single thiamine phosphoric acid to be standard.

Single thiamine phosphoric acid, ATP, PEP, NADH, metaphosphoric acid, trichoroacetic acid(TCA), cyanogen bromide and NaOH can derive from Sigma Chemicals. and utilize Schellenberger etc., (Hoppe-Seyler ' Z.Physiol.Chem. (1967) 348, method 501-505) is synthesized HMP, according to Leder etc., (1970) Enzymology method, 18A, the method for 166-167 is synthesized THZ-P.The HMP kinases is analyzed in embodiment 5 link coupled HMP kinases, THZ kinases and HMP-P kinases and the calibrating of TMP-PP enzymic activity and the calibrating of THZ kinases is analyzed

Follow the tracks of HMP to HMP-P and THZ conversion by detecting the ADP that forms simultaneously to THZ-P.Utilize pyruvate kinase and serum lactic dehydrogenase (reagent enzyme), detect in the presence of phosphoenolpyruvic acid (PEP) NADH to NAD ⁺Conversion, follow the tracks of the formation of ADP.Monitor at 340nm.Pyruvate kinase and PEP promote the regeneration of ATP by ADP.ATP is the substrate that HMP kinases and HMP-P kinases all need.The calibrating analysis buffer is for having added 10 mM MgCl ₂Buffer A.HMP-P kinases and TMP-PP enzyme

Analyze HMP-P kinases and TMP-PP enzyme for calibrating, substrate HMP-P and THZ-P need be provided.HMP-P and THZ-P are produced to conversion from the conversion of THZ-P to HMP-P and THZ by HMP in the same reaction mixture.If add NADH, utilize the calibrating of HMP kinases and THZ kinases to analyze and to follow the tracks of this conversion.When HMP transforms and THZ transforms to THZ-P and carries out (being respectively about 50mM and 20mM) when abundant, adding HMP-P kinases and TMP-PP enzyme to HMP-P.Produce back adding HMP-P kinases and TMP-PP enzyme at HMP-P and THZ-P, guarantee that the starting point concentration of these substrates in all reacting holes is constant.By Kawasaki etc., (1990) bacteriology magazine, 172, the method for 6145-6147 is identified the amount of the TMP that forms.After TMP produces time enough (general 15 minutes), stop enzyme reaction with metaphosphoric acid or TCA.Oxidation TMP under alkaline condition.Measure the fluorescence of gained thiachrome derivative with excitation wavelength 360+10nm and emission wavelength 430 ± 10nm.Calibration method

No matter analysis is all examined and determine with same procedure in the source of enzyme.In the 96 hole droplet plates of 300ml, examine and determine analysis.Total calibrating analytical reaction volume is 100ml.Mix substrate to scale and make that final concentration is (in the droplet plate): HMP (100mM), THZ (50 mM), ATP (5mM), PEP (1000 mM), and NADH (200 mM).The 10X substrate mixture can move in the 10ml/ hole.Also but mix reagent enzyme and HMP kinases are to add simultaneously.The recommended amounts of the ADP detection/reconstituted mixt of each reaction is 1.0 unit pyruvate kinases and 1.0 unit serum lactic dehydrogenases.This can be used as guidance, and according to experience adjustments enzyme amount.After HMP kinases and THZ kinase reaction are finished with about 5mM/ minute speed responsing (within 10-15 minute), add HMP-P kinases and TMP-PP enzyme.(by the activity decision of HMP-P kinases and TMP-PP enzyme) adds 50 mL 10% (w/v) metaphosphoric acids or 20% (w/v) trichoroacetic acid(TCA) (pH 1.4) termination reaction behind the certain intervals.Centrifugal culture plate, precipitating proteins moves into other droplet plate with supernatant liquor.The 0.3M cyanogen bromide that adds 50ml mixes, and adds the 1M NaOH of 100ml subsequently.Read the orifice plate value with excitation wavelength 360 ± 10nm and emission wavelength 430 ± 10nm by fluorophotometric droplet plate reader.Use single thiamine phosphoric acid to be standard.

Single thiamine phosphoric acid, ATP, PEP, NADH, metaphosphoric acid, trichoroacetic acid(TCA), cyanogen bromide and NaOH can derive from Sigma Chemicals.Utilize Schellenberger etc., (Hoppe-Seyler ' Z.Physiol.Chem. (1967) 348, method 501-505) is synthesized HMP, according to Leder etc., (1970) Enzymology method, 18A, the method for 166-167 is synthesized THZ-P.Embodiment 6 is by DNA reorganization vitro recombination HMP-P kinases/TMP-PP enzyme gene

As described in the embodiment 6 by proteic Arabidopis thaliana HMP-P kinases/TMP-PP enzyme gene before the pcr amplification coding.Basically handle the dna fragmentation that obtains as (1994) PNAS 91:10747-10751 as described in the Stemmer with the DNase I, from reaction mixture, remove the PCR primer.No primer carries out PCR reaction, is that the band primer carries out the PCR reaction subsequently, all as described in Stemmer et al. (1994) the PNAS 91:10747-10751.The gained dna fragmentation is cloned into pTRC99a (Pharmacia, Cat no:27-5007-01), utilize Biorad gene pulse instrument to be transformed into coli strain NI500 (genetics storage center by manufacturers's indication, New Haven, the U.S.) or coli strain NI400 (Nakayama and Hayashi (1972) bacteriology magazine, 112:1118-1126).The bacterium that growth transforms in the substratum that the inhibition that contains inhibition concentration exists selects the bacterium colony of growing when having inhibition to exist.The bacterium colony of picking growth in the presence of the inhibition of normal inhibition concentration, the purifying of ruling repeatedly.Its plasmid of purifying is determined to insert segmental dna sequence dna by the plasmid cDNA of this test.Embodiment 7 irregular extension method vitro recombination HMP-P kinases/TMP-PP enzyme genes

Separately the proteinic Arabidopis thaliana HMP-P kinases of encoding mature/TMP-PP enzyme gene and intestinal bacteria thiD are cloned into the polylinker of pBluescript carrier.Basically as Zhao etc., (1998) Nature Journal biotechnology fascicle, 16:258-261 is described, utilizes " reverse primer " and " M1320 primer " (Stratagene Catalog) to carry out PCR and reacts.With suitable Restriction Enzyme digest amplification PCR fragment, and be cloned into pTRC99a, the HMP-P kinases of sudden change/TMP-PP enzyme gene is as screening as described in the embodiment 6.B. separately the proteinic Arabidopis thaliana HMP-P kinases of encoding mature/TMP-PP enzyme gene and intestinal bacteria thiE are cloned into the polylinker of pBluescript carrier.Basically as Zhao etc., (1998) Nature Journal biotechnology fascicle, 16:258-261 is described, utilizes " reverse primer " and " M1320 primer " (Stratagene Catalog) to carry out PCR and reacts.With suitable Restriction Enzyme digest amplification PCR fragment, and be cloned into pTRC99a, the HMP-P kinases of sudden change/TMP-PP enzyme gene is as screening as described in the embodiment 6.C. separately the proteinic Arabidopis thaliana HMP-P kinases of encoding mature/TMP-PP enzyme gene and intestinal bacteria thiD and thiE are cloned into the polylinker of pBluescript carrier.Basically as Zhao etc., (1998) Nature Journal biotechnology fascicle, 16:258-261 is described, utilizes " reverse primer " and " M1320 primer " (Stratagene Catalog) to carry out PCR and reacts.With suitable Restriction Enzyme digest amplification PCR fragment, and be cloned into pTRC99a, the HMP-P kinases of sudden change/TMP-PP enzyme gene is as screening as described in the embodiment 6.The initial sum of embodiment 8 rice embryogenesis cells suspension is kept

With the prematurity small ear shelling with emulsus endosperm of Japonica rice varieties " Taipei 309 ", with 70% (v/v) ethanol surface sterilization 1 minute, 6% Losantin 20 minutes, it is inferior to give a baby a bath on the third day after its birth with sterile distilled water subsequently.(2,4-D), 0.35% agarose of pH5.8 solidifies the immature embryo of the last 28 ℃ of culture of isolated of MS substratum (Murashige and Skoog, 1962) containing 3% sucrose, 2mg/l 2,4 dichloro benzene ethoxyacetic acid.After one week, by transferring on the fresh culture separately and cultivate the callus material that produces by scutellum (scntella) weekly.After beginning for 4 weeks, 3-4 callus transferred to contain 20ml R2 substratum (R2 salt and VITAMIN [people such as Ohira, 1973], lmg/l 2,4-D, 500mg/l 2-morpholino ethane sulfonic acid [MES], 3% sucrose are in 50ml culturing bottle pH5.8).Culture under half-light 28 ℃ be stored on 220 rev/mins the rotary shaking table, weekly substratum is replaced with the fresh culture of equivalent.Select division fast, crisp callus, cultivate in the new container by the thin callus suspension of 2ml being transferred in the 20ml R2 substratum it is gone down to posterity.

The bombardment of embodiment 9 displaing microparticles

3-4 days 2-3 monthly age suspension culture of cultivation go down to posterity in advance as the target cell of bombarding.Preceding 4 hours of particle bombardment, about 500mg cell is solidified the plasmolysis substratum at 0.35% contained agarose of 5.5cm culture dish, and (R2 salt and VITAMIN, 1mg/l 2,4-D, 3% sucrose, 0.5M sucrose pH5.8) are gone up tiling and are the individual layer of 2cm diameter.Flow into rifle (Finer etc., 1992) with particle and send the gold grain (Aldrich Cat.#32,658-5, spherical gold powder 1.5-3.0 μ m) that DNA wraps quilt to embryo generation suspension cell.The particle bag is carried out as described in people such as Vain (1993) basically: 5 μ l equal portions plasmid solutions are assigned in the 0.5ml reaction tubes, placed on ice.Particle is suspended in 96% ethanol with 100mg/ml, vortex vibration 2 minutes.Ethanol is replaced with isopyknic aseptic ddH ₂Vibrate this suspension 1 minute of O, vortex.This washing step must repeat once.At last particle is resuspended to aseptic ddH with 100mg/ml ₂Among the O.In each DNA equal portions, add 25 μ l particle suspension, will manage vortex vibration 1 minute, immediately add 25 μ l aseptic, ice the CaCl of precooling ₂(2.5M is dissolved in ddH ₂O), continue vortex vibration 1 minute.(0.1M is dissolved in ddH to add the aseptic spermidine of 10 μ l ₂O), vortex vibration suspension placed 5 minutes on ice, therebetween solids precipitation once more.Shifting out 50 μ l does not have the particle supernatant liquor, and remaining suspension (15 μ l) is used to carry out 5 bombardments.Before each bombardment, particle need pass through violent pressure-vaccum resuspension.

Make cell cover the netted dividing plate of 500 μ m, be placed on and contain 14cm under the particulate filtration unit.In partial vacuum (2 * 10 ⁴Pa) down with 50 milliseconds single 8 bar pressure pulse release particles.

Embodiment 10: the conversion of wheat

A kind of optimization technique that wheat transforms comprises the particle bombardment to the prematurity wheat, and comprises high-sucrose or high malt sugar step before the gene delivery.Before bombardment, the embryo of arbitrary quantity (0.75-1mm is long) bed board is inoculated in and contains 3% sucrose (Murashige and Skoog, 1962) and 3mg/l 2, on the MS substratum of 4-D, carry out the body embryonal induction that can in the dark carry out.On selected bombardment same day, from inducing culture, take out embryo, place on the osmoticum (that is, general 15%, add the inducing culture of sucrose or maltose) with the concentration of hope.The idioplasm wall was separated 2-3 hour, then bombardment.Dull and stereotyped 20 embryos of each target are used always, but optional.With standard technique the suitable gene plasmid that contains is deposited on the big or small gold grain of micron.Parting pressure and use standard 80 mesh sieves with about 1000psi bombard each dull and stereotyped embryo with DuPont Biolistic helium device.After the bombardment, embryo is put back to the dark place recover about 24 hours (still on osmoticum).After 24 hours, take off embryo, put back on the inducing culture, before regeneration, placed about one month from osmoticum.After about one month, the embryo explants that forms embryo generation callus is transferred in the regeneration culture medium (MS+1mg/ rises NAA, and 5mg/ rises GA) that contains suitable selective agent in addition.After about one month, the seedling that forms is transferred in the big sterile chamber that is called as GA7s, wherein contained the selective agent of MS, 2% sucrose and the same concentrations of half amount intensity.Describe the stable conversion of wheat among the patent application EP0674715 in detail.The preparation of embodiment 11 corns (Zea mays) callus particular type elite inbred lines Funk2717

In the greenhouse, cultivate inbred lines Funk2717 maize plant to blooming self-pollination.The immature fringe that contains embryo that length is about 2-2.5mm shifts out from plant, and sterilization is 20 minutes in 10%Chlorox solution.Shift out embryo from seed asepsis, plate is sowed in containing 0.1mg/l2,4-D, 6% sucrose and 25mM L-proline(Pro) 0.24%Gelrite (initial medium) solidified OMS substratum by plumular axis court.27 ℃ in dark, cultivated for 2 weeks after, the callus that will form on scutellum is removed from embryo, plants plate in containing 0.5mg/l 2,4-D and with 0.24%Gelrite solidified B5 medium (Gamborg etc., 1968).Per two weeks are passaged to fresh culture with callus.After embryo being placed on the initial medium altogether for 8 weeks, identify the particular type of callus by the callus characteristic morphologic.Cross again after two months, callus is transferred to contains 2mg/l 2,4-D and go down to posterity with series on the Gelrite solidified N6 substratum and on this substratum.The preparation of embodiment 12 maize calli elite inbred lines Funk2717 suspension cultures

Above-mentioned callus was gone down to posterity 6 months at least altogether.The selected callus type that is used to go down to posterity be non-relatively phlegmatic temperament, particulate and be highly brittle a little less than, therefore it is separated into little individual cells aggregate once placing liquid nutrient medium.The culture that contains the aggregate of big amplifying cells does not keep.The about 500mg equal portions of maize calli particular type elite inbred lines Funk2717 are placed in the 125ml Delong flask, wherein contain 30ml band 2mg/l 2, the N6 substratum of 4-D.(130 rev/mins, 2.5cm rotates) 26 ℃ are cultivated a week down on rotary flask in the dark, replace substratum with fresh culture, and another all rear suspension liquids in this way go down to posterity once more.At that time, check culture, keep the culture that those do not show a large amount of amplifying cells.Abandon the suspension culture that contains big amplifying cells.Preferred tissue is made up of dense cytokinesis cell aggregation, it is characterized in that having than the more slick characteristic surface of common cell aggregation type.The culture that keeps has at least 50% with these little aggregates cell that is representative.This is the morphology of expectation.These suspension also have quick growth velocity, and the doubling time was less than for 1 week.By suspension culture that 0.5ml PCV immigration 25ml fresh culture is gone down to posterity weekly.Go down to posterity 4-6 after week with this method, and subculture increases 2-3 doubly weekly.Keep and wherein to surpass 75% cell and further go down to posterity for the culture of the morphology of expectation.Show that by total selection content wherein the flask of optimal morphology goes down to posterity, and keeps strain.Sometimes the stainless steel mesh periodic filtering that uses 630 microns per two weeks to be increasing the dispersion of culture, but optional.Embodiment 13 prepares protoplastis from the corn suspension culture

The 1-1.5ml PCV of above-mentioned suspension culture cell cultivates in the mixture of 10-15ml filtration sterilization, and mixture is by 4% cellulase RS and KMC salts solution (8.65 g/l KCl, 16.47g/l MgCl ₂X6H ₂O and 12.5 g/l CaCl ₂X2H ₂O, 5 g/l MES, pH 5.6) in 1%Rhozyme form.30 ℃ with slow shaking table on 3-4 hour time of digestion.Monitor the release for preparing protoplastis in the sample down in inverted microscope.The protoplastis that following collection discharges: prepare sample by 100 microns hole sizer net filtrations, use 50 microns hole sizer net filtrations subsequently.Pass through screen cloth with KMC salts solution washing protoplastis with enzyme solution initial volume equal volume.The 10ml protoplastis is prepared sample place each disposable plastic centrifuge tube, the 0.6M sucrose solution of lower berth 1.5-2ml (being buffered to pH 5.6) with 0.1%MES and KOH.In the centrifugal centrifuge tube of 60-100x g 10 minutes, utilize the liquid-transfering gun point at the protoplastis that is gathered into band at the interface, place fresh tube.The protoplastis goods that in the fresh KMC salts solution of 10ml, suspend again, in 60-100xg centrifugal 5 minutes.Remove supernatant liquor, discard, gentle resuspended protoplastis in the residue drop progressively adds the KMC solution of 13/14 intensity of 10ml.Behind the recentrifuge 5 minutes, supernatant liquor is removed, protoplastis is resuspended in the KMC solution of 6/7 intensity.Duplicate samples such as get and be used for counting, the centrifugal protoplastis that precipitates once more.With 10 ⁷/ ml is resuspended in protoplastis the KM-8p substratum or contains 6 mM MgCl ₂0.5M N.F,USP MANNITOL in, or in other suitable substratum, be used for the conversion described in the following embodiment.Transform and cultivate as following use protoplastis suspension.Embodiment 14 carries out corn by electroporation and transforms

A. the institute in steps (except heat-shocked) all carry out (22-28 ℃) in room temperature.Resuspended protoplastis is in containing 0.1%MES and 6mM MgCl in above-mentioned final step ₂0.5M N.F,USP MANNITOL in, in Dialog Electroporator chamber, measure the resistance of this suspension, and be adjusted to 1-1.2K; Utilize 300 mM MgCl ₂Solution.Immerse 45 ℃ of water-baths by the test tube that will contain sample and carried out the protoplastis heat-shocked in 5 minutes, subsequently in cooled on ice to room temperature.With 4 μ g contain just like the linearization plasmid of described plant selectivity hygromycin gene in 1987 such as Rothstein or as above-mentioned mosaic gene construct and 20 μ g tire ox thymus gland carrier DNAs add in the duplicate samples such as 0.25ml of this suspension.Contain 30 mM MgCl to the protoplastis adding ₂0.5M N.F,USP MANNITOL in 24%PEG solution (MW 8000) 0.125ml.Gentleness but thorough mixing mixture, incubation 10 minutes.Sample transfer is gone in the electroporation apparatus chamber, with 10 seconds spacing pulse samples 3 times, initial voltage 1500,1800,2300 or 2800V/cm, 10 milliseconds of exponential attenuation times.Following cultivation protoplastis.The sample room temperature is sowed plate in 6 centimetres of culture dish.

Following cultivation protoplastis.Under the room temperature sample is plated in the 6cm culture dish.After 5-15 minute, add and contain 1.2%SeaPlaque agarose and mg/l 2, the 3mlKM-8p substratum of 4-D.Thorough mixing agarose and protoplastis make substratum become gel.B. repeat aforesaid method with one or more following changes:

(1) with the resistance adjustment of protoplastis goods to 0.5-0.7K;

(2) PEG of Shi Yonging is the PEG with MW4000.

(3) do not add PEG, or add the 12%PEG of half volume.

(4) carry out pulse with 3 seconds intervals.

(5) behind the electroporation protoplastis is being planted plate in dish, placing on the dish that is cooled to 16 ℃ of room temperatures.

(6) after the electroporation step, protoplastis is placed test tube, (comprise 380 mg/l KCl, 18.375 g/l CaCl with the KMC solution of 6/7 intensity of 10ml or with W5 solution ₂X2H ₂O, 9g/l NaCl; 9g/l glucose, pH 6.0) washing, centrifugal 10 minutes then in 60xg, resuspended in 0.3ml KM substratum, as kind of plate as described in the A.

(7) do not add tire ox thymus gland carrier DNA.Embodiment 15 usefulness PEG handle the maize transformation protoplastis

A. in above-mentioned final step, protoplastis is resuspended in and contains 12-30 mM MgCl ₂The 0.5M mannitol solution.Carried out 45 ℃ of heat-shockeds 5 minutes as above-mentioned.Protoplastis is dispensed into is used in the equal portions that centrifuge tube transforms every pipe 0.3ml suspension protoplastis.Added in 10 minutes subsequently: (MW6000,40% contains 0.1MCa (NO for DNA and PEG solution ₃) ₂With 0.4M N.F,USP MANNITOL; Transfer to pH8-9 with KOH), final concentration reaches 20%PEG.To wait the gentle frequently jolting of duplicate samples, incubation 30 minutes then with protoplastis kind plate (each diameter 6cm culture dish 0.3ml initial native plastid suspension) in culture dish, is cultivated as described.

B. repeat this step, 30 minutes after scouring protoplastiss of incubation in above-mentioned PEG solution, method is to add 0.3ml W5 solution washing 5 times at interval with 2-3 minute.Centrifugal protoplastis suspension is removed supernatant liquor, cultivates protoplastis as described.

C. repeat above-mentioned steps, the PEG final concentration is modified between the 13-25%.Embodiment 16 is by the protoplast regeneration callus

The flat board that contains protoplastis in agarose places dark in 26 ℃.After 14 days, obtain colony from protoplastis.The agarose that will contain colony is transferred to the surface of 9cm diameter culture dish, and described culture dish contains band 2mg/l 2,4-D 0.24%Gelrite acidifying N6 substratum 30ml.This substratum is called 2N6.26 ℃ of cultured calli in dark, per two weeks are passaged to the callus small pieces on the fresh solid 2N6 substratum once more.The maize calli screening that embodiment 17 transforms

Embodiment above repeating in order to screen cell transformed, adds to the 2N6 substratum with 100mg/l or 200mg/l hygromycin B.The regeneration of embodiment 18 maize plants

A. callus is placed on the 2N6 substratum and keep, place ON6 (to contain the N6 substratum, do not contain 2,4-D) regenerate to cause with N61 substratum (contain the N6 substratum, contain 0.25mg/l 2,4-D and 10mg/l phytokinin).(with 16 hours/daylight of white fluorescent lamp with 840-8400 lx) cultivates the callus of growing on ON6 and the N61 substratum under illumination.The callus that to grow on the N61 substratum after two weeks is transferred to the ON6 substratum, is deleterious because prolong growth on the N61 substratum.The callus that goes down to posterity in per two weeks will be even callus will be transferred on the same medium prescription once more.Plantlet appears in about 4-8 week.In case plantlet has the 2cm height at least, it is transferred on the ON6 substratum of GA7 container.Form root in 2-4 week, when root fully forms when being enough to support growth, plantlet is moved into soil in the peat jar, in shade, grew in preceding 4-7 days.Usually back-off one transparent plastics cup was useful in 2-3 days on transplant, to help sclerosis.In case form plant, handle as normal maize plant, in the greenhouse, grow to maturation.In order to obtain progeny plant, self-pollination or hybridize with wild-type.

B. repeat the foregoing description, add 100mg/l or 200mg/l hygromycin B in the substratum for keeping callus.Embodiment 19 imports Schrock (Sorghum bicolor) protoplastis with DNA

Substantially as described in the top corn, the protoplastis of preparation Chinese sorghum suspension FS 562, last washing back in following solution with 10 ⁷/ ml is resuspended: 0.2 M N.F,USP MANNITOL, 0.1%MES, 72 mM NaCl, 70 mM CaCl ₂, 2.5 mM KCl, 2.5 mM glucose, regulating pH with KOH is 5.8, with 1.6-2 * 10 ⁶The density of/ml.With the 1ml equal portions protoplastis suspension is dispensed in the disposable tubule of plastics, adds 10 μ g DNA as described.Scope is in 6 when measuring between following electroporation apparatus 471 electrode devicies this solution resistance.In order to transform, DNA is added in the 10 microlitre sterile distilled waters, with as Paszkowski etc., 1984 described degerming.Gentle mixing solution, the pulse in room temperature (24-28 ℃) experience 400V/cm utilizes 471 electrode device BTX-Transfector300 electroporation apparatuss, and the attenuation index constant is 10ms.B. repeat above-mentioned steps, carry out following one or more modification: (1) working voltage is 200 Vcm-1, or between 100 Vcm-1 and 800 Vcm-1.(2) the exponential attenuation constant is 5 ms, 15 ms or 20 ms.The tire ox thymidine DNA that 50 μ g shear in (3) the 25 μ l sterilized waters adds with plasmid DNA.(4) use suitable Restriction Enzyme (as the BamH I) to handle before use, make the plasmid DNA linearizing.

Transform the back with 2 * 10 ⁶The density of/ml is incubated at protoplastis in the KM-8p substratum, does not add solidifying agent.Embodiment 20 imports DNA the protoplastis of Glycine max

As Tricoli etc., (1986) or Chowhury and Widholm (1985) or Klein etc., (1981) described method prepares Glycine max protoplastis.Basically as mentioned above DNA is imported these protoplastiss.As Tricoli etc., (1986) or Chowhury and Widholm (1985) or Klein etc., (1981) described cultivation protoplastis does not add alginic acid and solidifies substratum.

Above-mentioned disclosed embodiment is the property illustrated.Of the present inventionly openly will make those skilled in the art have various deformation of the present invention.All these conspicuous and foreseeable distortion are contained by appended claim.

＜110〉Novartis AG＜120〉＜130〉PH／5-30565／A／CGC2013＜140〉＜141〉＜150〉US 09／109，254＜151〉1998-06-30＜160〉7＜170〉PatentIn Ver.2.0＜210〉1＜211〉1569＜212〉DNA＜213〉＜220〉＜221〉CDS＜222〉 （ 1 ） .. （ 1566 ）＜223〉HMP-P／TMP-PPcDNA＜400〉1atg aat agc tta gga gga att agg agt tgg ccg gcg aat tgg aga agt 48Met Asn Ser Leu Gly Gty Ile Arg Ser Trp Pro Ala Asn Trp Arg Ser 1 5 10 15acg acg gcg tca atg acg acg acg gag agc gtt aga aag gta ccg caa 96Thr Thr Ala Ser Met Thr Thr Thr Glu Ser Val Arg Lys Val Pro Gln。

20 25 30gtt?tta?aca?gtg?gcg?gga?tca?gat?tcc?ggc?gcc?gga?gct?gga?att?caa 144Val?Leu?Thr?Val?Ala?Gly?Ser?Asp?Ser?Gly?Ala?Gly?Ala?Gly?Ile?Gln

35 40 45gcc?gac?ctt?aaa?gtc?tgc?gca?gct?cgt?ggt?gtg?tat?tgc?gct?tcc?gtc 192Ala?Asp?Leu?Lys?Val?Cys?Ala?Ala?Arg?Gly?Val?Tyr?Cys?Ala?Ser?Val

50 55 60ata?acc?gca?gtc?act?gct?cag?aac?act?cga?gga?gtt?caa?tct?gtt?cat 240Ile?Thr?Ala?Val?Thr?Ala?Gln?Asn?Thr?Arg?Gly?Val?Gln?Ser?Val?His?65 70 75 80ctt?crt?cct?ccg?gaa?ttt?arc?tct?gaa?cag?ctc?aaa?tcc?gtc?crc?tct 288Leu?Leu?Pro?Pro?Glu?Phe?Ile?Ser?Glu?Gln?Leu?Lys?Ser?Val?Leu?Ser

85 90 95gac?ttc?gaa?ttc?gac?gtc?gtg?aag?act?ggg?atg?ctt?cct?tct?act?gag 336Asp?Phe?Glu?Phe?Asp?Val?Val?Lys?Thr?Gly?Met?Leu?Pro?Ser?Thr?Glu

100 105 110atc?gtt?gag?gtt?ctt?ctt?caa?aat?cta?tca?gat?ttt?cca?gtt?cgt?gct 384Ile?Val?Glu?Val?Leu?Leu?Gln?Asn?Leu?Ser?Asp?Phe?Pro?Val?Arg?Ala

115 120 125ttg?gtt?gtt?gat?cct?gtg?atg?gta?tct?act?agt?ggt?cac?gtt?ttg?gct 432Leu?Val?Val?Asp?Pro?Val?Met?Val?Ser?Thr?Ser?Gly?His?Val?Leu?Ala

130 135 140ggt?tct?tct?att?ctc?tct?atc?ttt?aga?gag?aga?tta?cta?cca?att?gct 480Gly?Ser?Ser?Ile?Leu?Ser?Ile?Phe?Arg?Glu?Arg?Leu?Leu?Pro?Ile?Ala145 150 155 160gac?ata?att?acc?cca?aat?gtg?aaa?gag?gct?tct?gct?tta?ctt?gat?ggt 528Asp?Ile?Ile?Thr?Pro?Asn?Val?Lys?Glu?Ala?Ser?Ala?Leu?Leu?Asp?Gly

165 170 175ttt?cgg?att?gag?act?gtt?gca?gaa?atg?cgg?tct?gca?gca?aag?tcg?ttg 576Phe?Arg?Ile?Glu?Thr?Val?Ala?Glu?Met?Arg?Ser?Ala?Ala?Lys?Ser?Leu

180 185 190cat?gaa?atg?ggt?cct?aga?ttc?gta?ctt?gtt?aaa?ggt?ggt?gat?ctt?cct 624His?Glu?Met?Gly?Pro?Arg?Phe?Val?Leu?Val?Lys?Gly?Gly?Asp?Leu?Pro

195 200 205gac?tea?tca?gat?tca?gta?gat?gtt?tac?ttt?gat?ggc?aag?gag?ttt?cat 672Asp?Ser?Ser?Asp?Ser?Val?Asp?Val?Tyr?Phe?Asp?Gly?Lys?Glu?Phe?His

210 215 220gaa?ctc?cgt?tct?cct?cgc?ata?gct?aca?aga?aat?act?cat?ggg?act?ggt 720Glu?Leu?Arg?Ser?Pro?Arg?Ile?Ala?Thr?Arg?Asn?Thr?His?Gly?Thr?Gly225 230 235 240tgc?act?ttg?gct?tcc?tgt?att?gca?gct?gag?ctt?gca?aaa?ggc?tct?tcc 768Cys?Thr?Leu?Ala?Ser?Cys?Ile?Ala?Ala?Glu?Leu?Ala?Lys?Gly?Ser?Ser

245 250 255atg?ctc?tca?gcc?gtc?aag?gtg?gct?aaa?cgc?ttt?gtc?gat?aat?gcc?cta 816Met?Leu?Ser?Ala?Val?Lys?Val?Ala?Lys?Arg?Phe?Val?Asp?Asn?Ala?Leu

260 265 270gat?tac?agc?aaa?gat?att?gtc?att?ggc?agt?ggg?atg?caa?gga?cct?ttt 864Asp?Tyr?Ser?Lys?Asp?Ile?Val?Ile?Gly?Ser?Gly?Met?Gln?Gly?Pro?Phe

275 280 285gac?cat?ttt?ttt?ggt?ctt?aag?aag?gat?cct?caa?agt?tct?cga?tgc?agc 912Asp?His?Phe?Phe?Gly?Leu?Lys?Lys?Asp?Pro?Gln?Ser?Ser?Arg?Cys?Ser

290 295 300ata?ttc?aat?cca?gat?gac?ctg?ttt?cta?tat?gct?gtt?aca?gat?tct?aga 960Ile?Phe?Asn?Pro?Asp?Asp?Leu?Phe?Leu?Tyr?Ala?Val?Thr?Asp?Ser?Arg305 310 315 320atg?aac?aaa?aaa?tgg?aac?cgt?tcc?att?gtg?gat?gcc?ttg?aaa?gct?gct 1008Met?Asn?Lys?Lys?Trp?Asn?Arg?Ser?Ile?Val?Asp?Ala?Leu?Lys?Ala?Ala

325 330 335ata?gag?gga?ggg?gcc?acc?atc?ata?caa?ctg?agg?gag?aaa?gaa?gcc?gaa 1056Ile?Glu?Gly?Gly?Ala?Thr?Ile?Ile?Gln?Leu?Arg?Glu?Lys?Glu?Ala?Glu

340 345 350aca?cgg?gag?ttt?ctt?gaa?gaa?gca?aaa?gca?tgc?att?gat?ata?tgc?cgg 1104Thr?Arg?Glu?Phe?Leu?Glu?Glu?Ala?Lys?Ala?Cys?Ile?Asp?Ile?Cys?Arg

355 360 365tcc?cat?gga?gtt?agt?ttg?ctg?ata?aac?gac?agg?atc?gac?att?gcc?ctt 1152Ser?His?Gly?Val?Ser?Leu?Leu?Ile?Asn?Asp?Arg?Ile?Asp?Ile?Ala?Leu

370 375 380gct?tgt?gat?gct?gat?gga?gtc?cat?gtt?ggt?caa?tcc?gac?atg?ccg?gtt 1200Ala?Cys?Asp?Ala?Asp?Gly?Val?His?Val?Gly?Gln?Ser?Asp?Met?Pro?Val385 390 395 400gat?cta?gtt?cgg?tct?ctt?ctt?ggc?ccg?gac?aag?atc?ata?ggg?gtc?tca 1248Asp?Leu?Val?Arg?Ser?Leu?Leu?Gly?Pro?Asp?Lys?Ile?Ile?Gly?Val?Ser

405 410 415tgt?aag?aca?cca?gaa?caa?gct?cat?caa?gca?tgg?aaa?gat?ggt?gcg?gac 1296Cys?Lys?Thr?Pro?Glu?Gln?Ala?His?Gln?Ala?Trp?Lys?Asp?Gly?Ala?Asp

420 425 430tac?att?ggg?tca?gga?gga?gtt?ttt?cca?acg?aac?act?aag?gcc?aac?aat 1344Tyr?Ile?Gly?Ser?Gly?Gly?Val?Phe?Pro?Thr?Asn?Thr?Lys?Ala?Asn?Asn

435 440 445cgt?acc?ata?gga?ctt?gat?ggg?cta?aaa?gaa?gta?tgt?gaa?gca?tca?aaa 1392Arg?Thr?Ile?Gly?Leu?Asp?Gly?Leu?Lys?Glu?Val?Cys?Glu?Ala?Ser?Lys

450 455 460tta?ccg?gtt?gtt?gca?atc?gga?ggc?ata?ggg?atc?tca?aat?gct?ggg?tct 1440Leu?Pro?Val?Val?Ala?Ile?Gly?Gly?Ile?Gly?Ile?Ser?Asn?Ala?Gly?Ser465 470 475 480gtt?atg?cag?atc?gat?gca?ccg?aac?cta?aaa?ggt?gta?gca?gtt?gtg?tca 1488Val?Met?Gln?Ile?Asp?Ala?Pro?Asn?Leu?Lys?Gly?Val?Ala?Val?Val?Ser

485 490 495gct?ttg?ttc?gac?caa?gat?tgt?gtt?ttg?act?caa?gct?aag?aag?ttg?cat 1536Ala?Leu?Phe?Asp?Gln?Asp?Cys?Val?Leu?Thr?Gln?Ala?Lys?Lys?Leu?His

500 505 510aaa?acg?ctt?aaa?gag?agc?aaa?agg?gga?att?tga 1569Lys?Thr?Leu?Lys?Glu?Ser?Lys?Arg?Gly?Ile

515 520＜210〉2＜211〉522＜212〉PRT＜213〉Arabidopis thaliana＜400〉2Met Asn Ser Leu Gly Gly Ile Arg Ser Trp Pro Ala Asn Trp Arg Ser, 15 10 15Thr Thr Ala Ser Met Thr Thr Thr Glu Ser Val Arg Lys Val Pro Gln

20 25 30Val?Leu?Thr?Val?Ala?Gly?Ser?Asp?Ser?Gly?Ala?Gly?Ala?Gly?Ile?Gln

35 40 45Ala?Asp?Leu?Lys?Val?Cys?Ala?Ala?Arg?Gly?Val?Tyr?Cys?Ala?Ser?Val

50 55 60Ile?Thr?Ala?Val?Thr?Ala?Gln?Asn?Thr?Arg?Gly?Val?Gln?Ser?Val?His?65 70 75 80Leu?Leu?Pro?Pro?Glu?Phe?Ile?Ser?Glu?Gln?Leu?Lys?Ser?Val?Leu?Ser

85 90 95Asp?Phe?Glu?Phe?Asp?Val?Val?Lys?Thr?Gly?Met?Leu?Pro?Ser?Thr?Glu

100 105 110Ile?Val?Glu?Val?Leu?Leu?Gln?Asn?Leu?Ser?Asp?Phe?Pro?Val?Arg?Ala

115 120 125Leu?Val?Val?Asp?Pro?Val?Met?Val?Ser?Thr?Ser?Gly?His?Val?Leu?Ala

130 135 140Gly?Ser?Ser?Ile?Leu?Ser?Ile?Phe?Arg?Glu?Arg?Leu?Leu?Pro?Ile?Ala145 150 155 160Asp?Ile?Ile?Thr?Pro?Asn?Val?Lys?Glu?Ala?Ser?Ala?Leu?Leu?Asp?Gly

165 170 175Phe?Arg?Ile?Glu?Thr?Val?Ala?Glu?Met?Arg?Ser?Ala?Ala?Lys?Ser?Leu

180 185 190His?Glu?Met?Gly?Pro?Arg?Phe?Val?Leu?Val?Lys?Gly?Gly?Asp?Leu?Pro

195 200 205Asp?Ser?Ser?Asp?Ser?Val?Asp?Val?Tyr?Phe?Asp?Gly?Lys?Glu?Phe?His

210 215 220Glu?Leu?Arg?Ser?Pro?Arg?Ile?Ala?Thr?Arg?Asn?Thr?His?Gly?Thr?Gly225 230 235 240Cys?Thr?Leu?Ala?Ser?Cys?Ile?Ala?Ala?Glu?Leu?Ala?Lys?Gly?Ser?Ser

245 250 255Met?Leu?Ser?Ala?Val?Lys?Val?Ala?Lys?Arg?Phe?Val?Asp?Asn?Ala?Leu

260 265 270Asp?Tyr?Ser?Lys?Asp?Ile?Val?Ile?Gly?Ser?Gly?Met?Gln?Gly?Pro?Phe

275 280 285Asp?His?Phe?Phe?Gly?Leu?Lys?Lys?Asp?Pro?Gln?Ser?Ser?Arg?Cys?Ser

290 295 300Ile?Phe?Asn?Pro?Asp?Asp?Leu?Phe?Leu?Tyr?Ala?Val?Thr?Asp?Ser?Arg305 310 315 320Met?Asn?Lys?Lys?Trp?Asn?Arg?Ser?Ile?Val?Asp?Ala?Leu?Lys?Ala?Ala

325 330 335Ile?Glu?Gly?Gly?Ala?Thr?Ile?Ile?Gln?Leu?Arg?Glu?Lys?Glu?Ala?Glu

340 345 350Thr?Arg?Glu?Phe?Leu?Glu?Glu?Ala?Lys?Ala?Cys?Ile?Asp?Ile?Cys?Arg

355 360 365Ser?His?Gly?Val?Ser?Leu?Leu?Ile?Asn?Asp?Arg?Ile?Asp?Ile?Ala?Leu

370 375 380Ala?Cys?Asp?Ala?Asp?Gly?Val?His?Val?Gly?Gln?Ser?Asp?Met?Pro?Val385 390 395 400Asp?Leu?Val?Arg?Ser?Leu?Leu?Gly?Pro?Asp?Lys?Ile?Ile?Gly?Val?Ser

405 410 415Cys?Lys?Thr?Pro?Glu?Gln?Ala?His?Gln?Ala?Trp?Lys?Asp?Gly?Ala?Asp

420 425 430Tyr?Ile?Gly?Ser?Gly?Gly?Val?Phe?Pro?Thr?Asn?Thr?Lys?Ala?Asn?Asn

435 440 445Arg?Thr?Ile?Gly?Leu?Asp?Gly?Leu?Lys?Glu?Val?Cys?Glu?Ala?Ser?Lys

450 455 460Leu?Pro?Val?Val?Ala?Ile?Gly?Gly?Ile?Gly?Ile?Ser?Asn?Ala?Gly?Ser465 470 475 480Val?Met?Gln?Ile?Asp?Ala?Pro?Asn?Leu?Lys?Gly?Val?Ala?Val?Val?Ser

485 490 495Ala?Leu?Phe?Asp?Gln?Asp?Cys?Val?Leu?Thr?Gln?Ala?Lys?Lys?Leu?His

500 505 510Lys?Thr?Leu?Lys?Glu?Ser?Lys?Arg?Gly?Ile

515 520＜210〉3＜211〉1473＜212〉DNA＜213〉arabidopsis＜220〉＜221〉CDS＜222〉(1) .. (1470)＜223〉infer ripe mustard and belong to the cDNA coded sequence of albumen before HMP-P kinases/TMP-PP enzyme＜400〉3atg tta aca gtg gcg gga tca gat tcc ggc gcc gga gct gga att caa 48Met Leu Thr Val Ala Gly Ser Asp Ser Gly Ala Gly Ala Gly Ile Gln, 15 10 15gcc gac ctt aaa gtc tgc gca gct cgt ggt gtg tat tgc gct tcc gtc 96Ala Asp Leu Lys Val Cys Ala Ala Arg Gly Val Tyr Cys Ala Ser Val

20 25 30ata?acc?gca?gtc?act?gct?cag?aac?act?cga?gga?gtt?caa?tct?gtt?cat 144Ile?Thr?Ala?Val?Thr?Ala?Gln?Asn?Thr?Arg?Gly?Val?Gln?Ser?Val?His

35 40 45ctt?ctt?cct?ccg?gaa?ttt?atc?tct?gaa?cag?ctc?aaa?tcc?gtc?ctc?tct 192Leu?Leu?Pro?Pro?Glu?Phe?Ile?Ser?Glu?Gln?Leu?Lys?Ser?Val?Leu?Ser

50 55 60gac?ttc?gaa?ttc?gac?gtc?gtg?aag?act?ggg?atg?ctt?cct?tct?act?gag 240Asp?Phe?Glu?Phe?Asp?Val?Val?Lys?Thr?Gly?Met?Leu?Pro?Ser?Thr?Glu?65 70 75 80atc?gtt?gag?gtt?ctt?ctt?caa?aat?cta?tca?gat?ttt?cca?gtt?cgt?gct 288Ile?Val?Glu?Val?Leu?Leu?Gln?Asn?Leu?Ser?Asp?Phe?Pro?Val?Arg?Ala

85 90 95ttg?gtt?gtt?gat?cct?gtg?atg?gta?tct?act?agt?ggt?cac?gtt?ttg?gct 336Leu?Val?Val?Asp?Pro?Val?Met?Val?Ser?Thr?Ser?Gly?His?Val?Leu?Ala

100 105 110ggt?tct?tct?att?ctc?tct?atc?ttt?aga?gag?aga?tta?cta?cca?att?gct 384Gly?Ser?Ser?Ile?Leu?Ser?Ile?Phe?Arg?Glu?Arg?Leu?Leu?Pro?Ile?Ala

115 120 125gac?ata?att?acc?cca?aat?gtg?aaa?gag?gct?tct?gct?tta?ctt?gat?ggt 432Asp?Ile?Ile?Thr?Pro?Asn?Val?Lys?Glu?Ala?Ser?Ala?Leu?Leu?Asp?Gly

130 135 140ttt?cgg?att?gag?act?gtt?gca?gaa?atg?cgg?tct?gca?gca?aag?tcg?ttg 480Phe?Arg?Ile?Glu?Thr?Val?Ala?Glu?Met?Arg?Ser?Ala?Ala?Lys?Ser?Leu145 150 155 160cat?gaa?atg?ggt?cct?aga?ttc?gta?ctt?gtt?aaa?ggt?ggt?gat?ctt?cct 528His?Glu?Met?Gly?Pro?Arg?Phe?Val?Leu?Val?Lys?Gly?Gly?Asp?Leu?Pro

165 170 175gac?tca?tca?gat?tca?gta?gat?gtt?tac?ttt?gat?ggc?aag?gag?ttt?cat 576Asp?Ser?Ser?Asp?Ser?Val?Asp?Val?Tyr?Phe?Asp?Gly?Lys?Glu?Phe?His

180 185 190gaa?ctc?cgt?tct?cct?cgc?ata?gct?aca?aga?aat?act?cat?ggg?act?ggt 624Glu?Leu?Arg?Ser?Pro?Arg?Ile?Ala?Thr?Arg?Asn?Thr?His?Gly?Thr?Gly

195 200 205tgc?act?ttg?gct?tcc?tgt?att?gca?gct?gag?ctt?gca?aaa?ggc?tct?tcc 672Cys?Thr?Leu?Ala?Ser?Cys?Ile?Ala?Ala?Glu?Leu?Ala?Lys?Gly?Ser?Ser

210 215 220atG?ctc?tca?gcc?gtc?aag?gtg?gct?aaa?cgc?ttt?gtc?gat?aat?gcc?cta 720Met?Leu?Ser?Ala?Val?Lys?Val?Ala?Lys?Arg?Phe?Val?Asp?Asn?Ala?Leu225 230 235 240gat?tac?agc?aaa?gat?att?gtc?att?ggc?agt?ggg?atg?caa?gga?cct?ttt 768Asp?Tyr?Ser?Lys?Asp?Ile?Val?Ile?Gly?Ser?Gly?Met?Gln?Gly?Pro?Phe

245 250 255gac?cat?ttt?ttt?ggt?ctt?aag?aag?gat?cct?caa?agt?tct?cga?tgc?agc 816Asp?His?Phe?Phe?Gly?Leu?Lys?Lys?Asp?Pro?Gln?Ser?Ser?Arg?Cys?Ser

260 265 270ata?ttc?aat?cca?gat?gac?ctg?ttt?cta?tat?gct?gtt?aca?gat?tct?aga 864Ile?Phe?Asn?Pro?Asp?Asp?Leu?Phe?Leu?Tyr?Ala?Val?Thr?Asp?Ser?Arg

275 280 285atg?aac?aaa?aaa?tgg?aac?cgt?tcc?att?gtg?gat?gcc?ttg?aaa?gct?gct 912Met?Asn?Lys?Lys?Trp?Asn?Arg?Ser?Ile?Val?Asp?Ala?Leu?Lys?Ala?Ala

290 295 300ata?gag?gga?ggg?gcc?acc?atc?ata?caa?ctg?agg?gag?aaa?gaa?gcc?gaa 960Ile?Glu?Gly?Gly?Ala?Thr?Ile?Ile?Gln?Leu?Arg?Glu?Lys?Glu?Ala?Glu305 310 315 320aca?cgg?gag?ttt?ctt?gaa?gaa?gca?aaa?gca?tgc?att?gat?ata?tgc?cgg 1008Thr?Arg?Glu?Phe?Leu?Glu?Glu?Ala?Lys?Ala?Cys?Ile?Asp?Ile?Cys?Arg

325 330 335tcc?cat?gga?gtt?agt?ttg?ctg?ata?aac?gac?agg?atc?gac?att?gcc?ctt 1056Ser?His?Gly?Val?Ser?Leu?Leu?Ile?Asn?Asp?Arg?Ile?Asp?Ile?Ala?Leu

340 345 350gct?tgt?gat?gct?gat?gga?gtc?cat?gtt?ggt?caa?tcc?gac?atg?ccg?gtt 1104Ala?Cys?Asp?Ala?Asp?Gly?Val?His?Val?Gly?Gln?Ser?Asp?Met?Pro?Val

355 360 365gat?cta?gtt?cgg?tct?ctt?ctt?ggc?ccg?gac?aag?atc?ata?ggg?gtc?tca 1152Asp?Leu?Val?Arg?Ser?Leu?Leu?Gly?Pro?Asp?Lys?Ile?Ile?Gly?Val?Ser

370 375 380tgt?aag?aca?cca?gaa?caa?gct?cat?caa?gca?tgg?aaa?gat?ggt?gcg?gac 1200Cys?Lys?Thr?Pro?Glu?Gln?Ala?His?Gln?Ala?Trp?Lys?Asp?Gly?Ala?Asp385 390 395 400tac?att?ggg?tca?gga?gga?gtt?ttt?cca?acg?aac?act?aag?gcc?aac?aat 1248Tyr?Ile?Gly?Ser?Gly?Gly?Val?Phe?Pro?Thr?Asn?Thr?Lys?Ala?Asn?Asn

405 410 415cgt?acc?ata?gga?ctt?gat?ggg?cta?aaa?gaa?gta?tgt?gaa?gca?tca?aaa 1296Arg?Thr?Ile?Gly?Leu?Asp?Gly?Leu?Lys?Glu?Val?Cys?Glu?Ala?Ser?Lys

420 425 430tta?ccg?gtt?gtt?gca?atc?gga?ggc?ata?ggg?atc?tca?aat?gct?ggg?tct 1344Leu?Pro?Val?Val?Ala?Ile?Gly?Gly?Ile?Gly?Ile?Ser?Asn?Ala?Gly?Ser

435 440 445gtt?atg?cag?atc?gat?gca?ccg?aac?cta?aaa?ggt?gta?gca?gtt?gtg?tca 1392Val?Met?Gln?Ile?Asp?Ala?Pro?Asn?Leu?Lys?Gly?Val?Ala?Val?Val?Ser

450 455 460gct?ttg?ttc?gac?caa?gat?tgt?gtt?ttg?act?caa?gct?aag?aag?ttg?cat 1440Ala?Leu?Phe?Asp?Gln?Asp?Cys?Val?Leu?Thr?Gln?Ala?Lys?Lys?Leu?His465 470 475 480aaa?acg?ctt?aaa?gag?agc?aaa?agg?gga?att?tga 1473Lys?Thr?Leu?Lys?Glu?Ser?Lys?Arg?Gly?Ile

485 490＜210〉4＜211〉490＜212〉PRT＜213〉Arabidopis thaliana＜400〉4Met Leu Thr Val Ala Gly Ser Asp Ser Gly Ala Gly Ala Gly Ile Gln, 15 10 15Ala Asp Leu Lys Val Cys Ala Ala Arg Gly Val Tyr Cys Ala Ser Val

20 25 30Ile?Thr?Ala?Val?Thr?Ala?Gln?Asn?Thr?Arg?Gly?Val?Gln?Ser?Val?His

35 40 45Leu?Leu?Pro?Pro?Glu?Phe?Ile?Ser?Glu?Gln?Leu?Lys?Ser?Val?Leu?Ser

50 55 60Asp?Phe?Glu?Phe?Asp?Val?Val?Lys?Thr?Gly?Met?Leu?Pro?Ser?Thr?Glu?65 70 75 80Ile?Val?Glu?Val?Leu?Leu?Gln?Asn?Leu?Ser?Asp?Phe?Pro?Val?Arg?Ala

85 90 95Leu?Val?Val?Asp?Pro?Val?Met?Val?Ser?Thr?Ser?Gly?His?Val?Leu?Ala

100 105 110Gly?Ser?Ser?Ile?Leu?Ser?Ile?Phe?Arg?Glu?Arg?Leu?Leu?Pro?Ile?Ala

115 120 125Asp?Ile?Ile?Thr?Pro?Asn?Val?Lys?Glu?Ala?Ser?Ala?Leu?Leu?Asp?Gly

130 135 140Phe?Arg?Ile?Glu?Thr?Val?Ala?Glu?Met?Arg?Ser?Ala?Ala?Lys?Ser?Leu145 150 155 160His?Glu?Met?Gly?Pro?Arg?Phe?Val?Leu?Val?Lys?Gly?Gly?Asp?Leu?Pro

165 170 175Asp?Ser?Ser?Asp?Ser?Val?Asp?Val?Tyr?Phe?Asp?Gly?Lys?Glu?Phe?His

180 185 190Glu?Leu?Arg?Ser?Pro?Arg?Ile?Ala?Thr?Arg?Asn?Thr?His?Gly?Thr?Gly

195 200 205Cys?Thr?Leu?Ala?Ser?Cys?Ile?Ala?Ala?Glu?Leu?Ala?Lys?Gly?Ser?Ser

210 215 220Met?Leu?Ser?Ala?Val?Lys?Val?Ala?Lys?Arg?Phe?Val?Asp?Asn?Ala?Leu225 230 235 240Asp?Tyr?Ser?Lys?Asp?Ile?Val?Ile?Gly?Ser?Gly?Met?Gln?Gly?Pro?Phe

245 250 255Asp?His?Phe?Phe?Gly?Leu?Lys?Lys?Asp?Pro?Gln?Ser?Ser?Arg?Cys?Ser

260 265 270Ile?Phe?Asn?Pro?Asp?Asp?Leu?Phe?Leu?Tyr?Ala?Val?Thr?Asp?Ser?Arg

275 280 285Met?Asn?Lys?Lys?Trp?Asn?Arg?Ser?Ile?Val?Asp?Ala?Leu?Lys?Ala?Ala

290 295 300Ile?Glu?Gly?Gly?Ala?Thr?Ile?Ile?Gln?Leu?Arg?Glu?Lys?Glu?Ala?Glu305 310 315 320Thr?Arg?Glu?Phe?Leu?Glu?Glu?Ala?Lys?Ala?Cys?Ile?Asp?Ile?Cys?Arg

325 330 335Ser?His?Gly?Val?Ser?Leu?Leu?Ile?Asn?Asp?Arg?Ile?Asp?Ile?Ala?Leu

340 345 350Ala?Cys?Asp?Ala?Asp?Gly?Val?His?Val?Gly?Gln?Ser?Asp?Met?Pro?Val

355 360 365Asp?Leu?Val?Arg?Ser?Leu?Leu?Gly?Pro?Asp?Lys?Ile?Ile?Gly?Val?Ser

370 375 380Cys?Lys?Thr?Pro?Glu?Gln?Ala?His?Gln?Ala?Trp?Lys?Asp?Gly?Ala?Asp385 390 395 400Tyr?Ile?Gly?Ser?Gly?Gly?Val?Phe?Pro?Thr?Asn?Thr?Lys?Ala?Asn?Asn

405 410 415Arg?Thr?Ile?Gly?Leu?Asp?Gly?Leu?Lys?Glu?Val?Cys?Glu?Ala?Ser?Lys

420 425 430Leu?Pro?Val?Val?Ala?Ile?Gly?Gly?Ile?Gly?Ile?Ser?Asn?Ala?Gly?Ser

435 440 445Val?Met?Gln?Ile?Asp?Ala?Pro?Asn?Leu?Lys?Gly?Val?Ala?Val?Val?Ser

450 455 460Ala?Leu?Phe?Asp?Gln?Asp?Cys?Val?Leu?Thr?Gln?Ala?Lys?Lys?Leu?His465 470 475 480Lys?Thr?Leu?Lys?Glu?Ser?Lys?Arg?Gly?Ile

485 490＜210〉5＜211〉35＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: oligonucleotides aththiDEfor＜400〉5ggtattgagg gtcgcatgaa tagcttagga ggaat 35＜210〉6＜211〉43＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: oligonucleotides aththiDErev＜400〉6agaggagagt tagagcctca aattcccctt ttgctctctt taa 43＜210〉7＜211〉36＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: oligonucleotides aththiDEfor-ctp＜400〉7ggtattgagg gtcgcttaac agtggcggga tcagat 36

Claims (58)

1. encode and participate in the nucleotide sequence of the biosynthetic enzyme of VitB1 for one kind, wherein said enzyme has HMP-P kinase activity or TMP-PP enzymic activity.

2. the nucleotide sequence of claim 1, wherein said enzyme contains and the identical or similar basically aminoacid sequence of aminoacid sequence that is shown in SEQ ID NO:2.

3. the nucleotide sequence of claim 1, wherein said enzyme contains the aminoacid sequence that is shown in SEQ ID NO:2.

4. the nucleotide sequence of claim 1, wherein said nucleotide sequence is identical with the nucleotide sequence that is shown in SEQ IDNO:1 or similar basically.

5. the nucleotide sequence of claim 1, wherein said nucleotides sequence is shown in SEQ IDNO:1.

6. mosaic gene that contains the promotor that effectively is connected with the nucleotide sequence of claim 1.

7. recombinant vectors that contains the mosaic gene of claim 7, wherein said carrier can be transformed into host cell with being stabilized.

8. host cell that contains the carrier of claim 14, wherein said nucleotides sequence is listed in the described cell and can expresses.

9. one kind participates in the biosynthetic plant protein of VitB1, and wherein said protein has HMP-P kinase activity or TMP-PP enzymic activity.

10. the protein of claim 9, wherein said enzyme contains and the identical or similar basically aminoacid sequence of aminoacid sequence that is shown in SEQ ID NO:2.

11. the protein of claim 9, wherein said protein contains the aminoacid sequence that is shown in SEQ ID NO:2.

12. identify the inhibition plant-growth of chemical to be measured and the method for viability, comprise the steps: for one kind

(a) under its product synthetic condition of endonuclease capable catalysis, the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture is mixed with HMP-P kinase substrate or TMP-PP enzyme substrates;

(b) under the same conditions, chemical to be measured in second reaction mixture and enzyme are mixed with the substrate that is used for first reaction mixture, keep as the identical time in first reaction mixture;

(c) be determined at the activity of enzyme in first and second reaction mixtures; And

(d) when the activity of enzyme in second reaction mixture is starkly lower than enzyme in first reaction mixture active, select the inhibition plant-growth or the viability of chemical to be measured.

13. the method for claim 12, wherein said enzyme be by identical or similar basically with the nucleotide sequence that is shown in SEQ ID NO:1 nucleotide sequence coded, or have identical with the aminoacid sequence that is shown in SEQID NO:2 or similar basically aminoacid sequence.

14. the method for claim 12, the kinase whose substrate of wherein said HMP-P are 2-methyl-4-amino-5-oxymethylpyrimidine phosphoric acid (HMP-P).

15. the method for claim 12, the substrate of wherein said TMP-PP enzyme are 4-methyl-5-(beta-hydroxyethyl) thiazole phosphoric acid (THZ-P).

16. the method for claim 12, the substrate of wherein said TMP-PP enzyme are 2-methyl-4-amino-5-oxymethylpyrimidine tetra-sodiums (HMP-PP).

17. the method for claim 12, the activity of wherein said enzyme is measured by measuring the TMP that produces in the reaction mixture.

18. a method that suppresses non-expectation vegetation growth comprises step from the chemical of being identified by the method for claim 12 to the vegetation of non-expectation that use.

19. a kind of plant, vegetable cell, plant seed or plant tissue, it comprises the nucleotide sequence that coding has the enzyme of HMP-P kinase activity, wherein said nucleotide sequence is given plant, plant tissue, plant seed or a certain amount of chemical of being identified by the method for claim 12 of vegetable cell tolerance, described a certain amount of be the amount of HMP-P kinase activity in the inhibition wild-type plant under the normal circumstances.

20. plant that can obtain by the following method, comprise the parent who transforms plant or plant with isolated DNA molecule, wherein isolated DNA molecule comprises the nucleotide sequence of the enzyme of coding HMP-P kinase activity, and can in described plant, express nucleotide sequence, thereby give the tolerance of the chemical of this plant identifying by claim 12 method.

21. calibration method that comprises following steps:

(a) under its product synthetic condition of endonuclease capable catalysis, the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture is mixed with HMP-P kinase substrate or TMP-PP enzyme substrates;

(b) under the same conditions, chemical in second reaction mixture and enzyme-to-substrate are mixed, keep as the identical time in first reaction mixture;

(c) be determined at the activity of enzyme in first and second reaction mixtures; Wherein, if the link coupled enzymic activity is starkly lower than enzymic activity in first reaction mixture in second reaction mixture, then chemical can inhibitory enzyme activity.

22. identify the inhibition plant-growth of chemical to be measured and the method for viability, comprise the steps: for one kind

(a) under endonuclease capable catalysis TMP coupling synthetic condition, the enzyme that has the HMP kinase activity in first reaction mixture is mixed with HMP kinase substrate and TMP-PP enzyme substrates with the enzyme with HMP-P kinase activity or TMP-PP enzymic activity;

(b) under the same conditions, chemical in second reaction mixture and enzyme-to-substrate are mixed, keep as the identical time in first reaction mixture;

(c) be determined at the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first and second reaction mixtures; And

(d) when the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in second reaction mixture is starkly lower than the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture active, select the inhibition plant-growth or the viability of chemical to be measured.

23. the method for claim 22, the enzyme that wherein has HMP-P kinases or TMP-PP enzymic activity is from plant.

24. the method for claim 22, the enzyme that wherein has HMP-P kinases or a TMP-PP enzymic activity is by identical or similar basically with the nucleotide sequence that is shown in SEQ ID NO:1 nucleotide sequence coded, or has and the identical or similar basically aminoacid sequence of aminoacid sequence that is shown in SEQ ID NO:2.

25. the method for claim 22, wherein the kinase whose substrate of HMP is HMP.

26. the method for claim 22, wherein the substrate of TMP-PP enzyme is THZ-P.

27. the method for claim 22, the activity that wherein has the enzyme of HMP-P kinases or TMP-PP enzymic activity is measured by measuring the TMP that produces in the reaction mixture.

28. one kind with isolated DNA molecule plant transformed, vegetable cell, plant seed or plant tissue, wherein isolated DNA molecule comprises the nucleotide sequence that coding has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity, wherein said dna molecular is given plant, vegetable cell, plant seed or the plant tissue tolerance to a certain amount of chemical of being identified by claim 22 method, described a certain amount of be the amount of HMP-P kinase activity or TMP-PP enzymic activity in the inhibition plant under the normal circumstances.

29. plant that can obtain by the following method, comprise the parent who transforms plant or plant with isolated DNA molecule, wherein isolated DNA molecule comprises the nucleotide sequence that coding has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity, and can in described plant, express nucleotide sequence, thereby give the tolerance of the chemical of this plant identifying by claim 22 method.

30. calibration method that comprises the steps:

(a) under endonuclease capable catalysis TMP coupling synthetic condition, the enzyme that has the HMP kinase activity in first reaction mixture is mixed with HMP kinase substrate and TMP-PP enzyme substrates with the enzyme with HMP-P kinase activity or TMP-PP enzymic activity;

(b) under the same conditions, chemical in second reaction mixture and enzyme-to-substrate are mixed, keep as the identical time in first reaction mixture;

(c) be determined at the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first and second reaction mixtures;

Wherein, be starkly lower than the enzymic activity that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture if having the enzymic activity of HMP-P kinase activity or TMP-PP enzymic activity in second reaction mixture, then chemical can suppress to have the enzymic activity of the enzyme of HMP-P kinase activity or TMP-PP enzymic activity.

31. identify the inhibition plant-growth of chemical to be measured or the method for viability, comprise the steps: for one kind

(a) under endonuclease capable catalysis TMP coupling synthetic condition, the enzyme that has the THZ kinase activity in first reaction mixture is mixed with HMP-P kinase substrate and THZ kinase substrate with the enzyme with HMP-P kinase activity or TMP-PP enzymic activity;

(b) under the same conditions, chemical in second reaction mixture and enzyme-to-substrate are mixed, keep as the identical time in first reaction mixture;

(c) be determined at the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first and second reaction mixtures; And

(d) when the enzymic activity that has HMP-P kinase activity or TMP-PP enzymic activity in second reaction mixture is starkly lower than the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture active, select the inhibition plant-growth or the viability of chemical to be measured.

32. the method for claim 31, the enzyme that wherein has HMP-P kinase activity or TMP-PP enzymic activity is from plant.

33. the method for claim 31, the enzyme that wherein has HMP-P kinase activity or a TMP-PP enzymic activity is by identical or similar basically with the nucleotide sequence that is shown in SEQ ID NO:1 nucleotide sequence coded, or has and the identical or similar basically aminoacid sequence of aminoacid sequence that is shown in SEQ ID NO:2.

34. the method for claim 31, wherein the kinase whose substrate of HMP-P is HMP-P.

35. the method for claim 31, wherein the kinase whose substrate of THZ is THZ.

36. the method for claim 31, the activity that wherein has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity is measured by measuring the TMP that produces in the reaction mixture.

37. a method that suppresses non-expectation vegetation growth comprises step from the chemical of being identified by the method for claim 31 to the vegetation of non-expectation that use.

38. one kind with isolated DNA molecule plant transformed, vegetable cell, plant seed or plant tissue, wherein isolated DNA molecule comprises the nucleotide sequence that coding has the enzyme of HMP-P kinase activity and/or TMP-PP enzymic activity, wherein said dna molecular is given the tolerance of the chemical that plant, vegetable cell, plant seed or plant tissue identify a certain amount of method by claim 31, described a certain amount of be the amount of HMP-P kinase activity and/or TMP-PP enzymic activity in the inhibition plant under the normal circumstances.

39. plant that can obtain by the following method, comprise the step that transforms the parent of plant or plant with isolated DNA molecule, wherein isolated DNA molecule comprises the nucleotide sequence that coding has the enzyme of HMP-P kinase activity and/or TMP-PP enzymic activity, and can in described plant, express nucleotide sequence, thereby give the tolerance of the chemical of this plant identifying by claim 31 method.

40. calibration method that comprises the steps:

(a) under endonuclease capable catalysis TMP coupling synthetic condition, the enzyme that has the THZ kinase activity in first reaction mixture is mixed with THZ kinase substrate and HMP-P kinase substrate with the enzyme with HMP-P kinase activity or TMP-PP enzymic activity;

(b) under the same conditions, chemical in second reaction mixture and enzyme-to-substrate are mixed, keep as the identical time in first reaction mixture;

(c) be determined at the activity of the enzyme that has HMP-P kinase activity or TMP-PP enzymic activity in first and second reaction mixtures;

Wherein, be starkly lower than the enzymic activity that has HMP-P kinase activity or TMP-PP enzymic activity in first reaction mixture if having the enzymic activity of HMP-P kinase activity or TMP-PP enzymic activity in second reaction mixture, then chemical can suppress to have the activity of the enzyme of HMP-P kinase activity or TMP-PP enzymic activity.

41. an evaluation has the method for the chemical of herbicidal activity, wherein said chemical suppresses HMP-P kinases or TMP-PP enzymic activity in the plant, comprises the steps:

(a) obtain transgenic plant, plant tissue, plant seed or vegetable cell, wherein comprise the isolating nucleotide sequence that a kind of coding has HMP-P kinase activity or TMP-PP enzymic activity, and the enzymatic activity of energy overexpression HMP-P kinase activity or TMP-PP enzyme;

(b) with chemical application in transgenic plant, plant tissue, plant seed or vegetable cell, and isogenic non-conversion plant, vegetable cell, tissue or its part;

(c) growth or the survival behind mensuration transgenosis and non-conversion plant, vegetable cell, the organizations chemical;

(d) growth or the survival behind comparison transgenosis and non-conversion plant, vegetable cell, the organizations chemical;

Wherein, chemical suppresses the growth or the survival of transgenic plant, vegetable cell, tissue or its part, and the growth or the survival of the isogenic non-transgenic plant of not obvious inhibition, vegetable cell, tissue or its part.

42. the method for claim 41, the enzyme that wherein has HMP-P kinase activity or a TMP-PP enzymic activity is by identical or similar basically with the nucleotide sequence that is shown in SEQ ID NO:1 nucleotide sequence coded, or has and the identical or similar basically aminoacid sequence of aminoacid sequence that is shown in SEQ ID NO:2.

43. a method that suppresses non-expectation vegetation growth comprises the chemical that any method is identified in the vegetation of non-expectation is used by claim 22 or 41.

44. one kind with isolated DNA molecule plant transformed, vegetable cell, plant seed or plant tissue, wherein isolated DNA molecule comprises the nucleotide sequence that coding has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity, wherein said dna molecular is given the tolerance of the chemical that plant, vegetable cell, plant seed or plant tissue identify a certain amount of method by claim 41, described a certain amount of be the amount of HMP-P kinase activity or TMP-PP enzymic activity in the inhibition plant under the normal circumstances.

45. plant that can obtain by the following method, comprise the step that transforms the parent of plant or plant with isolated DNA molecule, wherein isolated DNA molecule comprises the nucleotide sequence that coding has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity, and can in described plant, express nucleotide sequence, thereby give the chemical resistance of this plant identifying by claim 41 method.

46. one kind is suppressed the method for weed growth in the field of the crop of the crop seed that contains plantation or plant selectivity, comprises the steps:

(a) plantation herbicide tolerant crop or crop seed, it is with isolated DNA molecule plant transformed or plant seed, wherein isolated DNA molecule comprises the nucleotide sequence that coding has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity, and wherein said nucleotide sequence can be expressed in described plant or plant seed; And

(b) use a certain amount of weedicide to the crop in field or crop seed and weeds, institute's consumption suppresses naturally occurring HMP-P kinase activity or TMP-PP enzymic activity, and wherein, weedicide suppresses the growth of weeds, and the growth of not obvious inhibition crop.

47. reorganization dna molecular, wherein said reorganization dna molecule encode has the HMP-P kinases of weedicide enhanced tolerance or TMP-PP enzyme, described weedicide suppresses HMP-P kinase activity or the TMP-PP enzymic activity by a kind of template DNA molecule encoding, and described reorganization dna molecular is derived by this template DNA molecule.

48. mutant DNA molecule that the template DNA molecule that has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity by reorganization coding obtains, wherein said mutant DNA molecule encoding has the HMP-P kinases of weedicide enhanced tolerance or TMP-PP enzyme, and described weedicide suppresses HMP-P kinase activity or the TMP-PP enzymic activity by this template DNA molecule encoding.

49. the template DNA molecule of an enzyme that has HMP-P kinase activity or TMP-PP enzymic activity from coding forms the method for mutant DNA molecule that coding has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity, wherein said template DNA molecule has been cut into double-stranded random fragment, and described method comprises the steps:

(a) add at least a strand or double chain oligonucleotide in the double-stranded random fragment of gained colony, wherein said oligonucleotide comprises a zone identical with the template DNA molecule and one and the allogenic zone of template DNA molecule;

(b) double-stranded random fragment of gained and oligonucleotide sex change are become single chain molecule;

(c) incubation under such condition with gained single chain molecule colony and polysaccharase, this condition can cause described single chain molecule to be annealed in described same area, right to form the annealing fragment, described same area is enough to make the annealing fragment, and a right side causes duplicating of the opposing party, forms a kind of double-stranded polynucleotide of sudden change thus;

(d) repeat the second and the 3rd step at least two circulations again, the gained mixture comprised that further circulation forms the double-stranded polynucleotide of further sudden change from the double-stranded polynucleotide of the sudden change in the third step that formerly circulates during wherein further round-robin second went on foot;

Wherein, the double-stranded polynucleotide encoding of sudden change has the HMP-P kinases of weedicide enhanced tolerance or TMP-PP enzyme, and described weedicide suppresses HMP-P kinase activity or the TMP-PP enzymic activity by this template DNA molecule encoding.

50. the method for claim 49, wherein a kind of template DNA molecule is from eukaryote.

51. the method for claim 49, wherein said eukaryote is a plant.

52. the method for claim 49, the kind of wherein said template DNA molecule is identical with nucleotide sequence shown in the SEQ IDNO:1 or similar basically.

53. the mutant DNA molecule that obtains by the method for claim 49, its coding has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity, the dna molecule encode of wherein said sudden change has the HMP-P kinases of weedicide enhanced tolerance or TMP-PP enzyme, and described weedicide suppresses HMP-P kinase activity or the TMP-PP enzymic activity by this template DNA molecule encoding.

54. at least two kinds of template DNA molecules inequality of an enzyme that has HMP-P kinase activity or TMP-PP enzymic activity from coding form the method for mutant DNA molecule that coding has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity, comprise the steps:

(a) add at least a oligonucleotide in the template DNA molecule, wherein said oligonucleotide comprises a zone identical with each template DNA molecule;

(b) sex change of gained mixture is become single chain molecule;

(c) incubation under such condition with gained single chain molecule colony and polysaccharase, this condition can cause oligonucleotide and the annealing of template DNA molecule, and the condition that wherein is used for the polymerization of polysaccharase is the polymerization product that obtains corresponding to the part of template DNA molecule;

(d) repeat the second and the 3rd step at least two circulations again, wherein the extension products that obtains in the 3rd step can cause the template DNA molecule be used for polymerization in next circulation; Form the double-stranded polynucleotide of sudden change thus, it comprises the sequence from the different templates dna molecular;

Wherein, the double-stranded polynucleotide encoding of sudden change has the HMP-P kinases of weedicide enhanced tolerance or TMP-PP enzyme, and described weedicide suppresses HMP-P kinase activity or the TMP-PP enzymic activity by this template DNA molecule encoding.

55. the method for claim 54, wherein a kind of template DNA molecule is from eukaryote.

56. the method for claim 54, wherein said eukaryote is a plant.

57. the method for claim 54, the kind of wherein said template DNA molecule is identical with nucleotide sequence shown in the SEQ IDNO:1 or similar basically.

58. the mutant DNA molecule that obtains by the method for claim 54, its coding has the enzyme of HMP-P kinase activity or TMP-PP enzymic activity, the dna molecule encode of wherein said sudden change has the HMP-P kinases of weedicide enhanced tolerance or TMP-PP enzyme, and described weedicide suppresses HMP-P kinase activity or the TMP-PP enzymic activity by this template DNA molecule encoding.