CN101166824A - Cloning of cytochrome P450 genes from nicotiana - Google Patents

Abstract

The present invention relates to P450 enzymes and nucleic acid sequences encoding P450 enzymes in Nicotiana, and methods of using those enzymes and nucleic acid sequences to alter plant phenotypes.

Description

The clone is from the cytochrome P450 gene of tobacco

The present invention relates to the nucleotide sequence of Codocyte cytochrome p 450 enzyme in tobacco (Nicotiana) plant (hereinafter being called P450 and P450 enzyme) and use these nucleotide sequences to change the method for plant phenotype.

Background

The enzyme reaction of the multiextent chemically different substrates of Cytochrome P450 catalysis comprises endogenous and oxidation, peroxidation and reductive metabolism the xenobiotic substrate.In plant, the biochemical route that P450 participates in comprises that plant product is synthetic, as class phenylpropyl alcohol (phenylpropanoid), alkaloid, terpenoid, lipid, cyanogenic glycoside and Glucosinolates (glycosinolate) (Chappel, Annu.Rev.Plant Physiol.Plant Mol.Biol.198,49:311-343).Cytochrome P450 is also referred to as P450-mercaptan reduced hematin, and usually as the terminal oxidase in the multicomponent electron transfer chain, this shifts chain and is called the monooxygenase system that contains P450.Special catalyzed reaction comprises that demethylation, hydroxylation, epoxidation, N-oxidation, sulfoxidation, N-, S-and O-take off the reduction of alkyl, desulfurization, deaminizating and nitrogen, nitro and N-oxide group.

The not same-action of tobacco plant P450 enzyme comprises the host who influences various plants metabolite such as class phenylpropyl alcohol, alkaloid, terpenoid, lipid, cyanogen glucosides, Glucosinolates and other chemical entities.In recent years, obviously some P450 enzymes can influence the plant metabolites composition of plant.For example, need to improve local flavor and the fragrance of certain plants for a long time, this is to change its selected distribution of fatty acids by breeding; Yet know little for the mechanism that participates in these leaf composition levels of control.Reduce the P450 enzyme relevant with fatty acid modifying and can promote gathering of desired fats acid, desired fats acid provides preferred leaf phenotype quality.The function of P450 enzyme and their extensive effects in plant is formed still remain to be found.For example, find the special P450 enzyme catalysis of class lipid acid resolve into unsettled C6-and C9-aldehyde and-alcohol, they are main contributors of fruit and plant " pure and fresh (fresh green) " smell.The P450 level that can change other new target is to improve the character that leaf is formed, and this is by modifying the lipid composite and relevant catabolite in the tobacco leaf.Some these class leaves compositions are influenced by aging, the old and feeble maturation that stimulates leaf quality character.Other report shows the functional effect of performance in changing lipid acid of P450 enzyme, and these lipid acid involved in plant-pathogenic agent interacts and disease resistance.

In other example, hint P450 enzyme participates in alkaloidal biosynthesizing.Nornicotine is the less important alkaloid of finding in the tobacco (Nicotianatabacuum).Suppose that its generation is the nicotine demethylation of regulating by P450, then in N position acidylate and nitrosylation, thereby produces a series of N-acyl group nornicotines (acylnonicotine) and N-nitrosonornicotine.Think that by the catalytic N-demethylation of P450 demethylase of inferring be the biosynthetic main source of nornicotine in the tobacco.Think that enzyme is MC, therefore do not have successful purifying nicotine demethylase up to now, also do not separate the gene that participates in.

In addition, suppose but the activity that do not prove the P450 enzyme is subjected to Genetic Control and also is subjected to influencing strongly of environmental factors.For example, when plant arrives the stage of maturity, think that the demethylation of nicotine significantly increases in the tobacco.In addition, think that demethylase gene comprises transposable element, can suppress the RNA translation when transposable element exists.

Before the present invention, the diversity of P450 enzyme, their different 26S Proteasome Structure and Functions make the research of tobacco P450 enzyme very difficult.In addition, clone's P450 enzyme to small part is hindered, because the localized protein of these films generally exists abundance low and usually for the purifying instability.Therefore, need P450 enzyme and the nucleotide sequence relevant in the plant identification with these P450 enzymes.In tobacco only special event several cytopigment tobacco P450 albumen.Invention described herein comprises has found a large amount of Cytochrome P450 fragments, they on its sequence identity basis corresponding to several groups of P450 kinds.

General introduction

The present invention relates to plant P450 enzyme.The invention further relates to plant P450 enzyme from tobacco.The present invention also relates in plant, express and be subjected to ethene and/or plant senescence inductive P450 enzyme.The present invention more relates to the nucleotide sequence that enzymic activity is arranged in the plant, for example oxygenase, demethylase etc. or other, and use these sequences so that the expression decreased of these enzymes or silence.Invention relates to the P450 enzyme of finding in the plant equally, and its contained nornicotine level is higher than the lower plant of nornicotine level.

On the one hand, invention relates to nucleotide sequence, and they are shown in SEQ.ID.1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145 and 147.

In second related fields, these nucleotide sequence identity are grouped above 75% fragment, and this depends on their regional identity, and terminator codon is arrived corresponding to first nucleic acid of Cytochrome P450 motif GXRXCX (G/A) back in the zone.Represent nucleic acid group and the various types of Table I that is shown in.

The third aspect, invention relates to aminoacid sequence, and they are shown in SEQ.ID.2,4,6,8,10,12,14,16,18,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146 and 148.

In the 4th related fields, these amino acid sequence identities are grouped above 71% fragment, and this depends on their regional identity each other, and terminator codon is arrived corresponding to first amino acid of Cytochrome P450 motif GXRXCX (G/A) back in the zone.Represented amino acid group and the various types of Table II that is shown in.

The 5th aspect of invention is to use nucleotide sequence, and they are shown in SEQ.ID.1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145 and 147.

In the 6th related fields, minimizing or the P450 enzyme of removing in the tobacco plant can temporarily be finished with the RNA viruses system.The assessment gained transforms or the phenotype of infection plant changes, include but not limited to analyze endogenous P450 rna transcription this, P450 expression of peptides and plant metabolism substrate concentration, use the common available technology of those of ordinary skills.

In the 7th importance, the present invention also relates to produce the transgene tobacco strain that the P450 enzyme activity level changes.According to invention, these transgenic strains comprise nucleotide sequence, and sequence effectively makes the expression decreased or the silence of some enzyme, thereby cause the phenotype effect in the tobacco.This nucleotide sequence comprises SEQ.ID.1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145 and 147.

In aspect inventing very important the 8th, the plant growing kind comprises the performance of reducing nucleic acid of the present invention, and the metabolite that can change the relative comparison plant distributes.

The 9th aspect the present invention relates to screen plant, more preferably tobacco, and contained gene of plant and professor's nucleotide sequence has significant nucleic acid identity.The advantage of using invention is to identify and select plant, and plant comprises the nucleotide sequence of the accurate or remarkable identity of tool, and wherein this kind of plant is the different floral part of routine or the transformed variety procedure of breeding, sudden change program or natural generation.Screening has the plant of remarkable identity to finish by assessment plant nucleic acid material, uses nucleic acid probe, and the bind nucleic acid detecting operation includes but not limited to nucleic acid hybridization and pcr analysis.Nucleic acid probe can be made up of professor's nucleotide sequence or its fragment, and they are corresponding to SEQ.ID.1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145 and 147.

The tenth aspect the present invention relates to the plant identification gene, more preferably tobacco, and corresponding professor's nucleotide sequence has significant amino acid identity.The genomic clone (genomic clans) that the evaluation of plant gene is comprised cDNA and those cDNA, genomic clone (preferably from tobacco) can be finished by screening plant cDNA library, nucleic acid probe and bind nucleic acid detecting operation are used in screening, and nucleic acid monitoring operation includes but not limited to nucleic acid hybridization and pcr analysis.Nucleic acid probe comprises nucleotide sequence or its fragment, and they are corresponding to SEQ.ID.1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145 and 147.

In aspect other the 11, can screen the cDNA expression library of expression of peptides, used antibody is at part or all of professor's aminoacid sequence.This aminoacid sequence comprises SEQ.ID.2,4,6,8,10,12,14,16,18,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146 and 148.

The accompanying drawing summary

Fig. 1 show nucleic acid SEQ.ID.NO.:1 and amino acid SEQ.ID.NO.:2.

Fig. 2 show nucleic acid SEQ.ID.NO.:3 and amino acid SEQ.ID.NO.:4.

Fig. 3 show nucleic acid SEQ.ID.NO.:5 and amino acid SEQ.ID.NO.:6.

Fig. 4 show nucleic acid SEQ.ID.NO.:7 and amino acid SEQ.ID.NO.:8.

Fig. 5 show nucleic acid SEQ.ID.NO.:9 and amino acid SEQ.ID.NO.:10.

Fig. 6 show nucleic acid SEQ.ID.NO.:11 and amino acid SEQ.ID.NO.:12.

Fig. 7 show nucleic acid SEQ.ID.NO.:13 and amino acid SEQ.ID.NO.:14.

Fig. 8 show nucleic acid SEQ.ID.NO.:15 and amino acid SEQ.ID.NO.:16.

Fig. 9 show nucleic acid SEQ.ID.NO.:17 and amino acid SEQ.ID.NO.:18.

Figure 10 show nucleic acid SEQ.ID.NO.:19 and amino acid SEQ.ID.NO.:20.

Figure 11 show nucleic acid SEQ.ID.NO.:21 and amino acid SEQ.ID.NO.:22.

Figure 12 show nucleic acid SEQ.ID.NO.:23 and amino acid SEQ.ID.NO.:24.

Figure 13 show nucleic acid SEQ.ID.NO.:25 and amino acid SEQ.ID.NO.:26.

Figure 14 show nucleic acid SEQ.ID.NO.:27 and amino acid SEQ.ID.NO.:28.

Figure 15 show nucleic acid SEQ.ID.NO.:29 and amino acid SEQ.ID.NO.:30.

Figure 16 show nucleic acid SEQ.ID.NO.:31 and amino acid SEQ.ID.NO.:32.

Figure 17 show nucleic acid SEQ.ID.NO.:33 and amino acid SEQ.ID.NO.:34.

Figure 18 show nucleic acid SEQ.ID.NO.:35 and amino acid SEQ.ID.NO.:36.

Figure 19 show nucleic acid SEQ.ID.NO.:37 and amino acid SEQ.ID.NO.:38.

Figure 20 show nucleic acid SEQ.ID.NO.:39 and amino acid SEQ.ID.NO.:40.

Figure 21 show nucleic acid SEQ.ID.NO.:41 and amino acid SEQ.ID.NO.:42.

Figure 22 show nucleic acid SEQ.ID.NO.:43 and amino acid SEQ.ID.NO.:44.

Figure 23 show nucleic acid SEQ.ID.NO.:45 and amino acid SEQ.ID.NO.:46.

Figure 24 show nucleic acid SEQ.ID.NO.:47 and amino acid SEQ.ID.NO.:48.

Figure 25 show nucleic acid SEQ.ID.NO.:49 and amino acid SEQ.ID.NO.:50.

Figure 26 show nucleic acid SEQ.ID.NO.:51 and amino acid SEQ.ID.NO.:52.

Figure 27 show nucleic acid SEQ.ID.NO.:53 and amino acid SEQ.ID.NO.:54.

Figure 28 show nucleic acid SEQ.ID.NO.:55 and amino acid SEQ.ID.NO.:56.

Figure 29 show nucleic acid SEQ.ID.NO.:57 and amino acid SEQ.ID.NO.:58.

Figure 30 show nucleic acid SEQ.ID.NO.:59 and amino acid SEQ.ID.NO.:60.

Figure 31 show nucleic acid SEQ.ID.NO.:61 and amino acid SEQ.ID.NO.:62.

Figure 32 show nucleic acid SEQ.ID.NO.:63 and amino acid SEQ.ID.NO.:64.

Figure 33 show nucleic acid SEQ.ID.NO.:65 and amino acid SEQ.ID.NO.:66.

Figure 34 show nucleic acid SEQ.ID.NO.:67 and amino acid SEQ.ID.NO.:68.

Figure 35 show nucleic acid SEQ.ID.NO.:69 and amino acid SEQ.ID.NO.:70.

Figure 36 show nucleic acid SEQ.ID.NO.:71 and amino acid SEQ.ID.NO.:72.

Figure 37 show nucleic acid SEQ.ID.NO.:73 and amino acid SEQ.ID.NO.:74.

Figure 38 show nucleic acid SEQ.ID.NO.:75 and amino acid SEQ.ID.NO.:76.

Figure 39 show nucleic acid SEQ.ID.NO.:77 and amino acid SEQ.ID.NO.:78.

Figure 40 show nucleic acid SEQ.ID.NO.:79 and amino acid SEQ.ID.NO.:80.

Figure 41 show nucleic acid SEQ.ID.NO.:81 and amino acid SEQ.ID.NO.:82.

Figure 42 show nucleic acid SEQ.ID.NO.:83 and amino acid SEQ.ID.NO.:84.

Figure 43 show nucleic acid SEQ.ID.NO.:85 and amino acid SEQ.ID.NO.:86.

Figure 44 show nucleic acid SEQ.ID.NO.:87 and amino acid SEQ.ID.NO.:88.

Figure 45 show nucleic acid SEQ.ID.NO.:89 and amino acid SEQ.ID.NO.:90.

Figure 46 show nucleic acid SEQ.ID.NO.:91 and amino acid SEQ.ID.NO.:92.

Figure 47 show nucleic acid SEQ.ID.NO.:93 and amino acid SEQ.ID.NO.:94.

Figure 48 show nucleic acid SEQ.ID.NO.:95 and amino acid SEQ.ID.NO.:96.

Figure 49 show nucleic acid SEQ.ID.NO.:97 and amino acid SEQ.ID.NO.:98.

Figure 50 show nucleic acid SEQ.ID.NO.:99 and amino acid SEQ.ID.NO.:100.

Figure 51 show nucleic acid SEQ.ID.NO.:101 and amino acid SEQ.ID.NO.:102.

Figure 52 show nucleic acid SEQ.ID.NO.:103 and amino acid SEQ.ID.NO.:104.

Figure 53 show nucleic acid SEQ.ID.NO.:105 and amino acid SEQ.ID.NO.:106.

Figure 54 show nucleic acid SEQ.ID.NO.:107 and amino acid SEQ.ID.NO.:108.

Figure 55 show nucleic acid SEQ.ID.NO.:109 and amino acid SEQ.ID.NO.:110.

Figure 56 show nucleic acid SEQ.ID.NO.:111 and amino acid SEQ.ID.NO.:112.

Figure 57 show nucleic acid SEQ.ID.NO.:113 and amino acid SEQ.ID.NO.:114.

Figure 58 show nucleic acid SEQ.ID.NO.:115 and amino acid SEQ.ID.NO.:116.

Figure 59 show nucleic acid SEQ.ID.NO.:117 and amino acid SEQ.ID.NO.:118.

Figure 60 show nucleic acid SEQ.ID.NO.:119 and amino acid SEQ.ID.NO.:120.

Figure 61 show nucleic acid SEQ.ID.NO.:121 and amino acid SEQ.ID.NO.:122.

Figure 62 show nucleic acid SEQ.ID.NO.:123 and amino acid SEQ.ID.NO.:124.

Figure 63 show nucleic acid SEQ.ID.NO.:125 and amino acid SEQ.ID.NO.:126.

Figure 64 show nucleic acid SEQ.ID.NO.:127 and amino acid SEQ.ID.NO.:128.

Figure 65 show nucleic acid SEQ.ID.NO.:129 and amino acid SEQ.ID.NO.:130.

Figure 66 show nucleic acid SEQ.ID.NO.:131 and amino acid SEQ.ID.NO.:132.

Figure 67 show nucleic acid SEQ.ID.NO.:133 and amino acid SEQ.ID.NO.:134.

Figure 68 show nucleic acid SEQ.ID.NO.:135 and amino acid SEQ.ID.NO.:136.

Figure 69 show nucleic acid SEQ.ID.NO.:137 and amino acid SEQ.ID.NO.:138.

Figure 70 show nucleic acid SEQ.ID.NO.:139 and amino acid SEQ.ID.NO.:140.

Figure 71 show nucleic acid SEQ.ID.NO.:141 and amino acid SEQ.ID.NO.:142.

Figure 72 show nucleic acid SEQ.ID.NO.:143 and amino acid SEQ.ID.NO.:144.

Figure 73 show nucleic acid SEQ.ID.NO.:145 and amino acid SEQ.ID.NO.:146.

Figure 74 show nucleic acid SEQ.ID.NO.:147 and amino acid SEQ.ID.NO.:148.

Figure 75 demonstration is used for the segmental process by PCR cloning of cytochrome P 450 cDNA.Show SEQ.ID.NO.149-156.

Figure 76 sets forth the amino acid identity of group number.

Describe in detail

Definition

Unless otherwise defined, whole technology used herein and scientific terminology and field ordinary skill people of the present invention The meaning that the member understands usually is identical. Singleton etc., (1994) " microorganism and molecular biosciences dictionary " (Dictionary of Microbiology and Molecular Biology), the 2nd edition, John Wiley and Sons (New York) provides the comprehensive dictionary of many terms used herein and gives the technical staff. As referred to herein It is for reference that whole patents and publication are included this paper in. For the object of the invention, define below following term.

" enzymatic activity " is used for comprising demethylation, hydroxylating, epoxidation, N-oxidation, sulfoxidation, N-, S-and O-Take off the reduction of alkyl, desulfurization, deaminizating and nitrogen, nitro and N-oxide group. Term " nucleic acid " refers to deoxidation Ribonucleotide or ribonucleotide polymer are with list or double chain form or justice or antisense, unless in addition limited System comprises the known analog of natural nucleotide, and the heterozygosis mode of itself and nucleic acid is similar to the nucleosides of natural generation Acid. Except as otherwise noted, specific nucleic acid sequence comprises its complementary series. Term " is operatively connected ", " can grasps Do in conjunction with " and " but with operating sequence " refer to the expression of nucleic acid control sequence (as promoter, burst or transcribe because of The arrangement of sub-binding site) with the 2nd kind of nucleotide sequence between functional the connection, wherein expression control sequenc impact is right Transcribed nucleic acid and/or the translation of 2 kinds of sequences of Ying Yudi.

When using about cell, term " recombinant " expression cell copies allos nucleic acid, express described nucleic acid or Expression of peptides, heterologous peptides or by the protein of allos nucleic acid coding. Recombinant cell can be expressed justice or antisense form Gene or genetic fragment, these forms are not found in n cell form (non-restructuring). Recombinant cell also can be shown Reach the gene of finding in the n cell form, but wherein gene is modified and is passed through again transfered cell of manual type.

" structural gene " is Gene Partial, comprises the DNA section of encoding proteins, polypeptide or its part, and removal pushes away 5 ' sequence of moving transcription initiation. The product that structural gene can be encoded in addition and can not translate. Structural gene can be thin Find seldom in the normal cell position that find or cell or its importing among the born of the same parents that it claims in the importing situation Be " heterologous gene ". Heterologous gene can all or part of acquisition from any source known in the art, comprise bacterium Genome or episome, the DNA of eucaryon, nuclear or plasmid DNA, cDNA, viral DNA or chemical synthesis. Structure Gene can comprise one or more to be modified, and modification can affect the biology of biologically active or its feature, expression product and live The property or chemical constitution, expression speed or express the mode of control. This modification includes but not limited to sudden change, inserts, The replacement of disappearance and one or more nucleotides. Structural gene can consist of continual coded sequence or it can comprise One or more intrones are by suitable montage circle combination. Structural gene can be translated maybe and can not translate, and comprises antisense Direction. Structural gene can be section combination, obtains from multiple source and gene order combination (natural generation or close Become, wherein the synthetic DNA that refers to chemical synthesis).

" obtain certainly " to be used in reference to and get, obtain, accept, follow the trail of, copy or hereditary certainly source (chemistry and/or biology). Generate derivative can by chemistry or biological operation (include but not limited to replace, add, insert, disappearance, extraction, Separate, suddenly change and copy) primary source.

" chemical synthesis " finger that relates to dna sequence dna is grouped into nucleotides in external assembling. Manual chemical synthesis DNA can use process (Caruthers, " method of DNA and RNA order-checking " (the Methodology of that better establishes DNA and RNA Sequencing), (1983), Weissman (volume), Praeger Publishers, New York, The 1st chapter) finishes; Robotics is synthetic can carry out with one of some commercial machines of buying.

Carrying out can be by local homology's algorithm of Smith and Waterman for relatively optimal sequence comparison, Adv.Appl.Math.2:482 (1981), the homology alignment algorithm by Needleman and Wunsch, J.Mol. Biol.48:443 (1970), the retrieval of similar method by Pearson and Lipman, Proc.Natl.Acad.Sci. (U.S.A.) 85:2444 (1988), by these algorithms (Wisconsin GAP in the Genetics software kit, BESTFIT, FASTA and TFASTA, Genetics Computer Group, 575 Science Dr., Madison, Wis.) computerization realize or undertaken by inspection.

The local basic gopher of sequence alignment (BLAST) of NCBI (Altschul etc., 1990) obtains to come from some The source comprises national biological information center (NCBI, Bethesda, Md.) and internet, is used for the binding sequence analysis Program blastp, blastn, blastx, tblastn and tblastx. It can Http:// www.ncbi.nlm.nih.gov/BLAST/ access. How to determine retouching of sequence homogeneity with this program State and to obtain from http://www.ncbi.nlm.nih.gov/BLAST/blast.help.html.

Be applied to amino acid sequence and term used herein " significant amino acid homogeneity " or " significant amino Acid sequence homogeneity " the expression peptide characteristic, wherein the sequence that comprises of peptide is compared with reference group, corresponding to cell Behind the pigment P450 motif GXRXCX (G/A) first amino acid have to the zone of translated polypeptide terminator codon to Few percent 70 sequence homogeneity, preferred percent 80 amino acid sequence identity, more preferably percent 90 amino acid sequence identity, most preferably 99 at least percent to 100 sequence homogeneity.

Be applied to nucleotide sequence and term used herein " significant nucleic acid homogeneity " or " significant nucleotide sequence Homogeneity " expression polynucleotide sequence feature, wherein the sequence that comprises of polynucleotides is compared with reference group, right Should be in first nucleic acid behind the Cytochrome P450 motif GXRXCX (G/A) to the zone of translated polypeptide terminator codon Have 75 at least percent sequence homogeneity, preferred percent 81 amino acid sequence identity, more preferably Percent 91 to 99 sequence homogeneity, most preferably 99 at least percent to 100 sequence homogeneity.

Nucleotide sequence significantly identical other indication is if 2 molecule each other hybridization under stringent condition. Strictly Condition depends on can be different under sequence and the varying environment. Generally, stringent condition is chosen as about 5 ℃ to about 20 ℃, Usually about 10 ℃ to about 15 ℃,, be lower than specific sequence at the heat fusion joint (Tm) of determining ionic strength and pH. Tm 50% target sequence and the temperature (under definite ionic strength and pH) of mating Probe Hybridization. Usually, stringent condition In salinity be about 0.02 mole and temperature at pH7 and be at least about 60 ℃. For example, assorted at standard Southern In the friendship process, stringent condition comprise the beginning in 6xSSC 42 ℃ wash, then exist in the temperature at least about 55 ℃ 0.2xSSC in wash in addition one or many, general about 60 ℃ and about 65 ℃ usually.

When polypeptide and/or their encoded protein matter are remarkable when identical, nucleotide sequence is also significantly identical, is used for the object of the invention.Therefore, during much at one polypeptide of a kind of nucleic acid sequence encoding and the 2nd kind of nucleotide sequence, these two kinds of nucleotide sequences are significantly identical, even they are because the degeneracy that genetic codon allows and not hybridizing under stringent condition (referring to Darnell etc., (1990) " molecular cell biology " (Molecular Cell Biology), the 2nd edition, Scientific American Books W.H.Freeman and Company New York are about the explanation of codon degeneracy and genetic code).Lipidated protein or homogeneity can as the polyacrylamide gel electrophoresis of protein sample, then be presented by dyeing by certain methods indication well known in the art.For some purpose, need high resolving power and use HPLC or similar purification process.

As used herein, term " carrier " uses about nucleic acid molecule, nucleic acid molecule with the DNA sector transfer in cell.Carrier function is in repetition DNA and can be independently duplicated in host cell.Term " media " uses with " carrier " exchange sometimes.As used herein, term " expression vector " refers to that recombinant DNA molecules, molecule comprise required encoding sequence and expresses in the specific host organism to be operatively connected the required suitable nucleotide sequence of encoding sequence.Expressing required nucleotide sequence in prokaryotic organism generally includes promotor, operon (choosing wantonly), often puts with the ribosome binding site of other sequence.The known genuine karyocyte uses promotor, enhanser, termination and polyadenylation signal.

The complete genetic engineering plant of root is arranged in order to regenerate, and nucleic acid can insert vegetable cell, for example by inoculation or any known vitro tissue culture technique in any technology such as the body with generate can be in complete plant the regenerated transformed plant cells.Therefore, for example inserting vegetable cell can cause a disease or non-pathogenic Agrobacterium tumefaciems (A.tumefaciens) by external inoculation.Also can use other this tissue culture technique.

" plant tissue " comprises differentiation and the indifferent tissue of plant, but is not limited to root, stem, leaf, pollen, seed, tumor tissues and multiple culturing cell form such as unicellular, protoplastis, embryo and callus.Plant tissue can be in plant or in organ, tissue or cell cultures.

As used herein, " vegetable cell " comprises the vegetable cell in the plant and the vegetable cell and the protoplastis of cultivation." cDNA " or " complementary DNA " refers generally to single strand dna, its nucleotide sequence and RNA complementary element.CDNA forms the RNA template action by ThermoScript II.

Obtain the method for nucleotide sequence

According to the present invention, RNA extracts from changing and the non-tobacco tissue that changes the tobacco strain.The RNA of Ti Quing is used to produce cDNA subsequently.Nucleotide sequence of the present invention then generates with 2 kinds of methods.

In first method, poly A rich RNA is extracted from plant tissue and cDNA by reverse transcription PCR.Strand cDNA is used to produce P450 specific PCR group subsequently, uses degenerated primer to add few d (T) reverse primer.Design of primers is based on the conservative motif of the height of P450.The example of specific degenerated primer is shown in Fig. 1.Further analyze the sequence fragment from plasmid, plasmid contains the insertion of suitable size.These sizes are inserted general ranges from about 300 to about 800 Nucleotide, and this depends on the primer.

In the second approach, initial construction a cDNA library.CDNA in the plasmid is used to produce P450 specific PCR group subsequently, uses degenerated primer to add T7 primer on the plasmid as reverse primer.As in the 1st kind of method, further analyze sequence fragment from plasmid, plasmid contains the insertion of suitable size.

The tobacco plant strain and the non-detectable plant lines of nornicotine level of the high-level nornicotine of known generation (transformation body) can be used as parent material.

Leaf can take out from plant and handle to activate the P450 enzymic activity of this paper definition with ethene then.Total RNA extracts with technology known in the art.The cDNA fragment produces with PCR (RT-PCR) and few d (T) primer subsequently, as described in Figure 1.But follow the construction cDNA library, this is more abundant description in the example of this paper.

P450 zymoid conserved regions can be as the template (Figure 75) of degenerated primer.Use degenerated primer, the special band of P450 can pass through pcr amplification.The band of the similar enzyme of indication P450 can be identified by dna sequencing.Determine PCR fragment feature can retrieve, compare with BLAST or other instrument to identify suitable candidate.

From identifying that fragments sequence information can be used for developing the PCR primer.These primers are used for making up quantification PCR primer from the RNA of the ethene processing plant tissue of transformation and non-transformation.Only be with (300-800bp) or the more highdensity band of tool to be used for further feature and determine that more the high-density indication changes the more high expression level in the strain from the suitable big or small DNA that changes strain.Carry out the reverse differences expression of analyzing with the whole clones of detection gained of extensive Southern.In invention in this respect, can carry out these extensive oppositely Southern and analyze, use from the total cDNA of the mark of different tissues and insert to screen all clones as probe with the hybridization of cloned DNA fragment.

On-radiation Northern trace is measured and also is used for determining the segmental feature of clone P450.

As above the nucleotide sequence of Jian Dinging can detect with the gene silent technology of virus induction (VIGS, Baulcombe, " the current viewpoint of plant biological " (Current Opinions in Plant Biology), 1999,2:109-113).

In invention on the other hand, RNA interfering technology (RNAi) is used for further determining the feature of tobacco plant cytochrome P 450 enzyme activity of the present invention.It is for reference that the reference of following this technology of description is included this paper in, Smith etc., Nature, 2000,407:319-320; Fir etc., Nature, 1998,391:306-311; Waterhouse etc., PNAS, 1998,95:13959-13964; Setalberg etc., Plant Molecular Biology, 1993,23:671-683; Baulcombe, " the current viewpoint of plant biological ", 1999,2:109-113; Brigneti etc., EMBO Journal, 1998,17 (22): 6739-6746.Plant can use RNAi technology, antisense technology or described multiple other method to transform.

Exist some to be used for the plant that exogenous genetic material is imported vegetable cell, obtains stable maintenance and express quiding gene.This technology comprises that accelerated packet is directly entered cell (United States Patent (USP) 4,945,050 of Cornell and DowElanco 5,141,131) by genetic material on particulate.Plant can be used the Agrobacterium technical transform, referring to the United States Patent (USP) 5,177,010 of University of Toledo; Texas A﹠amp; 5,104,310 of M; European patent application 0131624B1, european patent application 120516,159418B1; 176,112 of european patent application 120516,159418B1 and Schilperoot; United States Patent (USP) 5,149,645,5,469,976,5,464,763 and 4,940,838 and Schilperoot 4,693,976; The european patent application 116718,290799,320500 of MaxPlanck; The european patent application 604662 and 627752 of Japan Nicotiana; The european patent application 0267159,0292435 of Ciba Geigy and United States Patent (USP) 5,231,019; The United States Patent (USP) 5,463,174 and 4,762,785 of Calgene and the United States Patent (USP) 5,004,863 and 5,159,135 of Agracetus.Other transformation technology comprises whisker technology (whiskers technology), referring to the United States Patent (USP) 5,302,523 and 5,464,765 of Zeneca.Electroporation technology also is used to transform plant, referring to the WO 87/06614 of Boyce ThompsonInstitute, and 5,742,869,5,384,253 of Dekalb, the WO9209696 of PGS and WO9321335.All these transform patent and publication is included in for reference.Except many technology that are used to transform plant, the types of organization of contact foreign gene also can change.This tissue includes but not limited to that embryo is organized, callus I and II type, plumular axis, meristem etc.Nearly all plant tissue can dedifferente middle conversion, the appropriate technology that use technology personnel grasp.

But the exogenous genetic material that imports plant comprises selective marker.To the judgement that preferably is the technician of specific markers, but but but any following selective marker can be unlisted but use as any other gene that selective marker can be brought into play function with this paper.This marks packets of selecting is drawn together but is not limited to encode to the aminoglycoside phosphotransferase gene (Aph II) of the transposon Tn5 of microbiotic kantlex, Xin Meisu and G418 resistance and coding to glyphosate; Totomycin; Methotrexate; Phosphinothricin (bar); Imidazolone, sulphur urea and triazolo pyrimidine weedicide such as chlorosuluron; The gene of resistance such as bromoxynil, dalapon or tolerance.

But, need the operation report gene except selective marker.In some cases, but but operation report gene and do not have selective marker.Reporter gene is the gene that does not generally have or be expressed in receptor biological body or tissue.Reporter gene is encoded usually provides the protein of some phenotypes variations or enzyme characteristic.The example of this gene is provided in K.Weising etc., Ann.Rev.Genetics, and 22,421 (1988), it is for reference that it includes this paper in.Preferred hard-core glucuronidase (GUS) gene and the GFP gene of comprising of reporter gene.

In case the importing plant tissue, the available any means known in the art of the expression of structure gene are measured, and expression can be measured as the gene silencing amount (referring to U.S. Patent number 5,583,021, it is for reference to include this paper in) of mRNA, synthetic protein or the generation of transcribing.Technology becomes known for the extracorporeal culturing plant tissue, in some cases, is used in complete plant regeneration (EP application number 88810309.0).The process of the expression complex body that imports being transferred to commercial useful Cultivar is known to those skilled in the art.

In case obtain to express the vegetable cell of required P450 enzyme level, plant tissue and complete plant can regenerate thus, use method well known in the art and technology.Aftergrowth can be transferred to other strain and Cultivar by the conventional plant breeding technique by ordinary method regeneration and quiding gene subsequently.

Following example elaboration is finished the method for invention and is interpreted as illustrating and unrestricted invention scope, and invention scope defines in accessory claim.

Embodiment

Embodiment 1: plant tissue is grown and ethene is handled

Plant-growth

Plant is inoculated in the basin and 4 weeks of growth in the greenhouse.4 all big seedlings are transplanted in the independent basin and were grown 2 months in the greenhouse.Plant is watered water 2 times every day in growth, and water contains 150ppm NPK fertilizer.The unfolded greenery separate with plant to carry out following ethene to be handled.

Clone 78379

Tobacco strain 78379 is uncle's Lay taro leaf strains of being delivered by University of Kentucky (University of Kentucky), as the plant material source.Press tobacco cultivation field type culture 100 strain plants and transplant and mark with different numbers (1-100).Fertilising and field management as recommendation are carried out.

3/4ths are transformed into nornicotine with 20 and 100% nicotine in the 100 strain plants.1/4th make the nicotine less than 5% be transformed into nornicotine in the 100 strain plants.Plant numbers 87 changes minimum (2%) and plant numbers 21 and changes 100%.Transformation is non-transformation body less than 3% plant classification.Self-pollination seed and hybridization (21 * 87 and 87 * 21) seed of plant numbers 87 and plant numbers 21 are used to study heredity and phenotypic difference.From the plant of self-pollination 21 is to change body, and 99% is non-transformation body from 87 self-pollination body.Low change (5-15%) of other 1% demonstration from 87.Plant from reciprocal cross all is to change body.

Clone 4407

Tobacco strain 4407 is the uncle's Lay taro leaf strains as the plant material source.Select unified and representational plant (100) and mark.97 strains are that non-transformation body and 3 strains are to change body in the 100 strain plants.Plant number 56 transformation amounts minimum (1.2%) and plant number 58 transformation levels the highest (96%).Self-pollination seed and reciprocal cross seed obtain with 2 kind of plant.

Plant from self-pollination-58 separates with non-transformation body ratio with about 3: 1 transformation body.58-33 and 58-25 are accredited as respectively to isozygoty and change body and non-transformation body plant lines.The stable transformation of 58-33 is by analyzing its next son for determining.

The ethene treating processes

Greenery separate from 2-3 month plant of greenhouse growth and spray with 0.3% vinyl solution (Prep board vincofos (Rhone-Poulenc)).Each sprays leaf and is suspended from clothes hanger, and clothes hanger is equipped with humidifier and plastic covered is arranged.In processing, the sample leaf regularly sprays with vinyl solution.Ethene was handled the back about 24-48 hour, collected leaf and was used for the RNA extraction.Get another kind of small sample and be used for the metabolism component analysis to determine leaf metabolite concentration and more specific formation interested such as multiple alkaloid.

For example, the alkaloid analysis can followingly be carried out.Sample (0.1g) extracts solution with 0.5ml 2N NaOH, 5ml and vibrates at 150rpm, and extraction solution contains quinoline and the methyl t-butyl ether as internal standard.Sample is analyzed on HP 6890 GC that fid detector is housed.250 ℃ temperature is used for detector and syringe.HP post (30m-0.32nm-1m) is made up of fused quartz, and quartzy crosslinked with 5% phenol and 95% methyl silicon, post uses with 10 ℃ of per minutes 110-185 ℃ thermograde.Post is 100 ℃ of operated in flow rate with 1.7cm3 minute-1, and the division ratio is 40: 1, and volume injected is 2.1, and helium is as vector gas.

Embodiment 2:RNA separates

Extract for RNA, handle with described ethene from the medium leaf of 2 months big greenhouse growing plants.0 and 24-48 hour sample be used for RNA and extract.In some cases, the leaf sample under the aging course is taken from and is removed the plant of head inflorescence after 10 days.These samples also are used for extracting.(Valencia California) follows manufacturer's operation and separates total RNA for Qiagen, Inc. with the small-sized test kit of Rneasy plant.

Tissue sample is ground to fine powder under liquid nitrogen, the mortar and the pestle that use DEPC to handle.The tissue that about 100mg pulverizes is transferred to aseptic 1.5ml centrifuge tube.This sample hose places liquid nitrogen up to having collected all samples.Subsequently, the 450 μ l damping fluid RLT (being added with beta-mercaptoethanol) that provide in the test kit add each single pipe.The abundant vortex of sample was also hatched 3 minutes at 56 ℃.Lysate was applied to the QIAshredder column spinner that places the 2-ml collection tube then, with high speed centrifugation 2 minutes.Stream that collection is passed through and 0.5 volume ethanol add in the limpid lysate.Sample mixes preferably and transfers to the little column spinner of the Rneasy that places the 2-ml collection tube.Sample is with 10, centrifugal 1 minute of 000rpm.Then, 700 μ l damping fluid RW1 are drawn onto on the Rneasy post in the new collection tube and with 10, centrifugal 1 minute of 000rpm.Add once more damping fluid RPE to the Rneasy column spinner and with high speed centrifugation 2 minutes with desciccator diaphragm.Carry for removing any ethanol, film places independent collection tube and with the most centrifugal 1 minute.The Rneasy post is transferred to new 1.5ml collection tube, and the water that 40 μ l do not have the RNA enzyme directly is drawn onto on the Rneasy film.This whole wash-out pipe is with 10, centrifugal 1 minute of 000rpm.The property quality and quantity of total RNA is by sex change glutol and spectrophotometer analysis.

Poly (A) RNA follows manufacturer's operation with Oligotex poly A RNA purification kit (Qiagen Inc.) and separates.Use the total RNA of about 200 μ g in the 250 μ l maximum volumes.The damping fluid OBB of 250 μ l volumes and 15 μ lOligotex suspensions add the total RNA of 250 μ l.By the suction thorough mixing inclusion and on heat block 70 ℃ hatched 3 minutes.Sample placed room temperature about 20 minutes then.The oligotex:mRNA complex body precipitated by high speed centrifugation in 2 minutes.Except 50 μ l suspensions, all take out from micro-centrifuge tube.Sample is further handled with the OBB damping fluid.The oligotex:mRNA precipitation is resuspended to 400 μ l damping fluid OW2 by vortex.This mixture is transferred to the little column spinner that places new pipe and with high speed centrifugation 1 minute.Column spinner is transferred to new pipe and is added 400 μ l damping fluid OW2 in addition in post.Pipe was with high speed centrifugation 1 minute subsequently.Column spinner is transferred to final 1.5ml micro-centrifuge tube.Sample is with 60 μ l heat (70C) damping fluid OEB wash-outs.Poly A product is by sex change glutol and spectrophotometer analysis.

Embodiment 3: reverse transcription-PCR

Article 1, the cDNA chain is followed manufacturer's operation (Invitrogen, Carlsbad, California) generation with the SuperScript ThermoScript II.Poly A rich RNA/oligo dT primer mixture is made up of the total RNA that is less than 5 μ g, 1 μ l10mM dNTP mixture, 1 μ l widow (dT) 12-18 (0.5 μ g/ μ l) and as many as 10 μ l DEPC-treating water.Each sample was hatched 5 minutes at 65 ℃, placed then at least 1 minute on ice.The preparation feedback mixture adds following each composition by order: 2 μ l 10X RT damping fluids, 4 μ l 25mM MgCl2,2 μ l 0.1M DTT and 1 μ l remove the recombinant RNA enzyme inhibitors of RNA enzyme.Add 9 μ l reaction mixtures, be drawn onto on each RNA/ primer mixture and gentle the mixing.It was hatched 2 minutes and each pipe of 1 μ l Super Script II RT adding at 42 ℃.Pipe was hatched 5 minutes at 42 ℃.Be reflected at 72 ℃ and stop 15 minutes and freezing on ice.Sample is by centrifugal collection, and 1 μ l RNA enzyme H adds each pipe and hatched 20 minutes at 37 ℃.(degenerated primer among Figure 75 is SEQ.ID.149-156) with 100 picomole reverse primers (mixture of 18 few d of Nucleotide (T) then is 1 base at random) with 200 picomole forward primers to finish the 2nd PCR.

Reaction conditions be 94 ℃ 2 minutes, carry out 40 PCR circulation subsequently: 94 ℃ 1 minute, 45 to 60 ℃ 2 minutes, 72 ℃ 3 minutes, 72 ℃ were extended 10 minutes in addition.

Amplification sample microlitre by electrophoresis with 1% agarose gel analysis.The fragment of correct size is from the agarose gel purifying.

Embodiment 4: produce the PCR segment group

From the PCR fragment of embodiment 3 follow manufacturer explanation be connected in the pGEM-T Easy carrier (Promega, Madison, Wisconsin).The connection product is transformed into the JM109 competent cell and cultivates on the LB flat board and is used for indigo plant/white selection.Select bacterium colony and 37 ℃ of overnight growth in 96 orifice plates that 1.2ml LB substratum is arranged.Produce the bacterium colony that refrigerated storage liquid is used for all selections.Plasmid DNA Beckman Biomeck 2000miniprep robot technology and Wizard SV Miniprep test kit (Promega) purifying from flat board.Plasmid DNA is with 100 μ l water elutions and be stored in 96 orifice plates.Size is measured and inserted to plasmid with EcoR1 digestion and by 1% agarose gel analysis to determine DNA.Contain plasmid CEQ 2000 sequenators (Beckman, Fullerton, California) order-checking that 400-600bp inserts.Sequence is compared by the BLAST retrieval with the GenBank database.P-450 associated clip and further analysis have been identified.

Embodiment 5: make up the CDNA library

The cDNA library handles from ethene by following that the total RNA of preparation makes up the leaf.At first, total RNA extracts from the ethene of tobacco strain 58-33 and handles leaf, uses acid phenol and the chloroform extraction operation revised.Retouching operation is to use 1 gram tissue, and tissue is ground and extracts damping fluid (100mM Tris-HCl, pH8.5 at 5ml subsequently; 200mM NaCl; 10mM EDTA; 0.5SDS) mesoscale eddies, damping fluid adds 5ml phenol (pH5.5) and 5ml chloroform.Centrifugal extraction sample is preserved supernatant.This extraction step repeats 2-3 time again and presents limpid up to supernatant.Add about 5ml chloroform to remove Determination of Trace Phenol.RNA is from mixing the supernatant partly precipitated, and this is by adding 3 times of volume ETOH and 1/10 volume 3M NaOAc (pH5.2) and preserving 1 hour at-20 ℃.After transferring to the Corex Glass Containers, it 4 ℃ with 9, centrifugal 45 minutes of 000RPM.Precipitation is washed with 70% ethanol, 4 ℃ with 9,000RPM rotated 5 minutes.After the precipitation drying, sedimentary RNA is dissolved in the water that 0.5ml does not have the RNA enzyme.The property quality and quantity of total RNA is respectively by sex change glutol and spectrophotometer analysis.

The total RNA of gained separates poly A+RNA by following operation, uses the operation of few d (T) Mierocrystalline cellulose (Invitrogen) and little centrifugal column spinner (Invitrogen).The total RNA of about 20mg carries out 2 purifying to obtain high quality poly A+RNA.Analyze poly A+RNA product, by the sex change glutol and subsequently the known full-length gene of RT-PCR to guarantee high quality mRNA.In addition, handle and change body leaf and ethene and handle the poly A+RNA that changes the body leaf and carry out Northern and analyze, use total length p450 as probe to handle non-transformation body leaf, zero hour ethene from ethene.(KPL RNA detector Northern trace test kit, Gaithersburg are the basis Maryland), and each sample uses 1.8 μ g poly A+RNA in the operation that method provides with manufacturer's explanation.The RNA that the contains glue transfer of spending the night uses 20X SSC as transfering buffering liquid.

Secondly, poly A+RNA as template to generate the cDNA library, use cDNA synthetic agent box, ZAP-cDNA synthetic agent box, ZAP-cDNA Gigapack III gold clone test kit (Stratagene, La Jolla, California).Method comprises manufacturer's operation of following detailed description.About 8 μ g poly A+RNA are used for the construction cDNA library.Analyze elementary library and show about 2.5 * 10 ⁶-1 * 10 ⁷Pfu.The quality background test in library is measured by complementation and is finished, and uses IPTG and X-gal, and the spot of wherein recombinating is to be higher than 100 times of expression of background response.

Show that by random PCR quantitative analysis library the mean size that inserts cDNA is about 1.2kb.Method is used two following step PCR method.For the 1st step, the design reverse primer is to obtain from the segmental preliminary sequence information of P450.The reverse primer of design and T3 (forward) primer are used for from the corresponding gene of cDNA amplified library.Agarose electrophoresis is carried out in PCR reaction, and corresponding high-molecular weight band is cut, purifying, clone and order-checking.In the 2nd step, clone to obtain total length P450 with the PCR that reverse primer (from P450 3 ' UTR design) is used from subsequently from the new primer of P450 5 ' UTR or start code district design as forward primer.

The P450 fragment generates from the cDNA library that makes up by pcr amplification, as described in embodiment 3, except the reverse primer difference.Be positioned at cDNA and insert the T7 primer (see figure 5) of downstream plasmid as reverse primer.The PCR fragment is separated, clone and order-checking, as described in embodiment 4.

Expection those skilled in the art can make many modifications and variations according to the detailed description of foregoing invention in the practice invention.As a result, these modifications and variations are included in the following claim scope.

Embodiment 6: feature-reverse SOUTHERN engram analysis of determining cloned sequence

All P450 clones that identify in the previous example are carried out the extensive oppositely southern engram analysis of on-radiation to detect differential expression.The expression level of observing between different P450 bunches is very different.Carrying out to high expression level further detects in real time.

Following the carrying out of on-radiation southern trace process.

1) total RNA extracts from ethene and handles transformation body (58-33) and non-transformation body leaf, uses Qiagen Rnaeasy test kit, as described in embodiment 2.

2) probe generates by vitamin H end mark strand cDNA, and cDNA obtains from the poly A of top step rich RNA.Producing this mark strand cDNA is to change body and the total RNA of non-transformation body (Invitrogen) by RT-PCR, as described in embodiment 3, except using biotinylated few dT as primer (Promega); These are used as probe to hybridize with cloned DNA.

3) plasmid DNA digests with Restriction Enzyme EcoR1 and the race agarose gel.Glue while drying also goes to 2 nylon membranes (Biodyne B).1 film with change the body probe hybridization, 1 film and non-transformation body probe hybridization in addition.UV-cross linking membrane before the hybridization (automatically cross-linked setting, 254nm, Stratagene, Stratalinker).

In addition, insert, use to be positioned at the sequence of p-GEM plasmid, T3 and SP6 two arms as primer from each plasmid pcr amplification.The PCR product is by running 96 holes ready-made (ready-to-run) agarose gel analysis.The insertion point of confirming is on 2 nylon membranes.1 film with change the body probe hybridization, 1 film and non-transformation body probe hybridization in addition.

4) Hybond membrane and follow manufacturer explanation and wash, revised the severity of washing (Enzo Diagnostics, Inc, Farmingdale, NY).Film with hybridization buffer (2xSSC cushion methane amide, contains washing agent and hybridizes toughener) 42 ℃ of prehybridizations 30 minutes and with 10 μ l sex change probes 42 ℃ of hybridization.Washed 1 time 10 minutes and washed 4 times 15 minutes with caudacoria room temperature in 1X hybridization lavation buffer solution at 65 ℃.Film can be used for detecting.

5) detect the film washed, this is by alkali phosphatase enzyme mark, then the NBT/BCIP colorimetric (? colometric) detect, as described in manufacturer's testing process (Enzo Diagnostics, Inc.).Film was washed 3 times 10 minutes with the 1X detection reagent with 1x lock solution room temperature sealing 1 hour, washed 2 times 5 minutes with the pre-color reaction damping fluid of 1x, and trace 30-45 minute of developing the color in chromophoric solution then is up to appearance point.All reagent by manufacturer provide (Enzo Diagnost ics, Inc.).

In some cases, (Gibco test kit, Carlsbad California) from non-transformation body (58-25) with change on total RNA that body (58-33) is and carry out, use to be specific to the segmental primer of P-450 1 step RT-PCR.Relatively RT-PCR is following carries out:

1) extracts the total RNA that handles transformation body (58-33) and non-transformation body (58-25) leaf from ethene, as described in embodiment 2.

2) poly (A) RNA from total RNA extracts with the Qiagen test kit, as described in embodiment 2.

3) 1 step RT-PCR follows manufacturer's process (Invitrogen) and carries out with the primer that is specific to clone P-450.Poly A rich RNA adds reaction mixture, being 25 μ l 2X reaction mixtures, 1 μ l, 10 μ M sense primers, 1 μ l, 10 μ M antisense primers, 1 μ l RT/Platinum taq mixture and mending water to 50 μ l therewith.Reaction conditions be 50 ℃ 20 minutes, subsequently 94 ℃ 2 minutes, carry out 40 PCR circulation: 94 ℃ 30 seconds, 55 ℃ 30 seconds, 70 ℃ 1 minute, 72 ℃ were extended 10 minutes in addition.10 microlitres amplifications sample by electrophoresis with 1% agarose gel analysis.

Embodiment 7: feature-NORTHERN engram analysis of determining cloned sequence

Except the Southern engram analysis, hybridize some films and as described in the example of Northern engram analysis, detect.Northern hybridization is used for the mRNA of following detection tobacco differential expression.

The 1st step, probe preparation: the random primer method be used for from clone p450 dna fragmentation prepare probe (the biotinylated test kit of random primer DNA, KPL).Mix following ingredients: 0.5 μ gDNA template (in water-bath, boiled 5-10 minute and use before freezing) on ice; 1X random primer solution; 1X dNTP mixture; 10 unit K lenow and water add so that reaction reaches 50 μ l.Mixture was hatched 1-4 hour at 37 ℃.Reaction stops with 2 μ l 200mM EDTA.Probe by before use 95 ℃ hatch and came sex change in 5 minutes.

In the 2nd step, specimen preparation: the RNA specimen preparation is from the fresh leaf and the old and feeble leaf of ethene processing and non-processing.In some cases, use poly A rich RNA.About total RNA of 15 μ g or 1.8 μ g mRNA (extracting method of RNA and mRNA is described in embodiment 5) become equal-volume with DEPC H2O (5-10 μ l).Sample-loading buffer (the 1x MOPS that adds equal volume; 18.5% formaldehyde; 50% methane amide; 4%Ficoll400; Tetrabromophenol sulfonphthalein) and 0.5 μ lEtBr (0.5 μ g/ μ l).Sample was 90 ℃ of heating 5 minutes, and is freezing on ice.

In the 3rd step, by electrophoretic separation RNA: sample carries out electrophoresis on glutol (1% agarose, 1x MOPS, 0.6M formaldehyde), with 1XMOP damping fluid (0.4M morpholino propane sulfonic acid; 0.1M sodium acetate-3xH2O; 10mMEDTA; Transfer to pH7.2 with NaOH).RNAs goes to Hybond-N+ film (nylon, Amersham PharmaciaBiotech), and this is at 10X SSC damping fluid (1.5M NaCl by the kapillary method; 0.15 shift Trisodium Citrate).Have film UV-before hybridization of RNA sample crosslinked (automatically cross-linked setting, 254nm, Stratagene, Stratalinker).

The 4th step, hybridization: film 5-10ml prehybridization damping fluid (5xSSC; 50% methane amide; 5xDenhardt solution; 1%SDS; The non-homogeneous DNA that 100 μ g/ml thermally denatures are sheared) at 42 ℃ of prehybridization 1-4 hour.Discard old prehybridization damping fluid, add new prehybridization damping fluid and probe.Hybridization is spent the night at 42 ℃ and is finished.Film was washed 15 minutes with the 2xSSC room temperature, then washed 2 times at 65 ℃ with 2xSSC, 0.1%SDS, washed with 0.1xSSC at last, or washed (choosing wantonly) with 0.1 SDS again at 65 ℃.

In the 5th step, detect: AP-streptavidin and CDP-Star are used to detect hybridization signal (KPL DNA detection device Northern trace test kit).Film sealed 30 minutes with 1X detector lock solution room temperature.Discarding sealing damping fluid and film is having 1: 10, and incubated at room is 1 hour in the new 1X detector lock solution of 000AP-SA.Film is washed 3 times in 1X Phosphoric acid esterase washing lotion, then measures damping fluid with the 1X Phosphoric acid esterase and washes 2 times.Signal detects with the CDP-Star chemical luminous substrate.Wet film exposes to X-ray film under Sha's human relations TM packing film.Analytical results and record.

The principal focal point of invention is to have found new gene, and gene can be handled and imported or bring into play in tobacco leaf character with in constituting keying action by ethene.As shown in the table, the Northern trace is used for determining non-relatively inducing plant, and which gene is handled by ethene and induced.What is interesting is that not all fragment institute in changing body and non-transformation body is influenced similar.Part checks order interested Cytochrome P450 fragment to determine their structural dependence.This information is used for separating subsequently and the full-length gene clone that checks order.Functional analysis with decreasing method is carried out in the complete plant of fragment gene is arranged.

Embodiment 8: the nucleic acid identity and the structural dependence of separating acid fragment

Surpassing 100 clone P450 fragments checks order to determine their structural dependence in conjunction with the Northern engram analysis.Method therefor uses forward primer, and primer is to be positioned near one of 2 common P450 motifs the P450 gene C-terminal.Forward primer is corresponding to Cytochrome P450 motif FXPERF or GRRXCP (A/G), as shown in Figure 1.Reverse primer uses the standard primer from plasmid or poly A tract portion, and plasmid SP6 or T7 are positioned at pGEM plasmid two arms.Used operation is as described below.

Spectrophotometry is used to estimate initial double-stranded DNA concentration, follows manufacturer's operation (BeckmanCoulter).Template is diluted with water to suitable concn, and 95 ℃ of heating came sex change in 2 minutes, with being placed on ice.Sequencing reaction is being prepared on ice, uses 0.5 to 10 μ l denatured DNA template, 2 μ l, 1.6 picomole forward primers, 8 μ l DTCS Quick Start Master mixtures, and the cumulative volume water adds to 20 μ l.The thermal cycling program is made up of 30 following circulations: 96 ℃ 20 seconds, 50 ℃ 20 seconds, 60 ℃ 4 minutes, then remain in 4 ℃.

Terminator sequence is by adding 5 μ l stop buffers (equal-volume 3M NaOAc and 100mM EDTA and 1 μ l20mg/ml glycogen).Sample was with cold 95% ethanol sedimentation of 60 μ l and at 6000g centrifugal 6 minutes.Abandon ethanol.Precipitation is washed with 70% cold ethanol of 200 μ l.After the precipitation drying, add 40 μ l SLS solution and resuspension precipitation.Cover one deck mineral oil.Sample places on CEQ 8000 automatic sequencers and is used for further analysis subsequently.

Be the check nucleotide sequence, nucleotide sequence uses the forward primer in relative P450 gene FXPERF or GRRXCP (A/G) district and the reverse primer of relative plasmid or poly A tract portion with 2 directions preface of resurveying.All order-checkings are carried out 2 times to the aspect at least with 2.

The mutual relatively coding region of Cytochrome P450 fragment nucleotide sequence, coding region corresponding to the district of coding GRRXCP (A/G) motif after first nucleic acid to terminator codon.Select of the indication of this district as genetic diversity between P450 albumen.Observe the different P450 gene of a large amount of heredity and be similar to the gene that other plant belongs to, surpass 70 genes.Comparing on the nucleotide sequence, finding that gene can place different sequence set on its sequence identity basis.It is 75% or higher sequence (being shown in Table I) that the best distinct packet of finding P450 member is defined as nucleic acid identity.Reduce identity per-cent and produce significantly bigger group.To nucleic acid identity be 81% or higher sequence observe preferred grouping, more preferably be grouped into 91% nucleic acid identity or higher, most preferably be grouped into 99% nucleic acid identity or higher.Most of group comprises at least 2 members and often is 3 or a plurality of member.Do not repeat to find other, the method that hint adopts can be separated the low and high expression level mRNA in the used tissue.

On 75% nucleic acid identity or higher basis, find that 2 Cytochrome P450 groups comprise the nucleotide sequence identity of relative front tobacco cell chromogene, gene is different in heredity between tobacco cell chromogene and group.Group 23 shows the nucleic acid identity with the GI:14423327 (or AAK62346) of the GenBank sequence GI:1171579 (CAA64635) of front Czernic etc. and Ralston etc. respectively, in the used parameter of Table I.GI:1171579 and the nucleic acid identity scope from 96.9% to 99.5% of organizing 23 members, and the identity scope from 95.4% to 96.9% that GI:14423327 organizes therewith.Organize the nucleic acid identity scope from 76.7% to 97.8% of the GenBank sequence GI:14423327 (AAK62346) of reports such as 31 members and Ralston.Do not have the P450 identity group of other table 1 to comprise the parameter identity of relative tobacco P450s gene, used as table 1, tobacco P450s gene is by Ralston etc., Czernic etc., Wang etc. or LaRosa and Smigocki report.

Shown in Figure 76, can obtain the consensus sequence of the suitable nucleic acid degeneracy of tool probe, be used to organize with preferred evaluation and the other member who separates from each group of tobacco plant.

Table I: tobacco P450 nucleotide sequence identity group

The group fragment

1 D58-BG7(SEQ?ID?NO.：1)，D58-AB1(SEQ?ID?NO.：3)，D58-BE4(SEQ?ID?NO.：7)

2 D56-AH7(SEQ?ID?NO.：9)；D13a-5(SEQ?ID?NO.：11)

3 D56-AG10(SEQ?ID?NO.：13)；D35-33(SEQ?ID?NO.：15)；D34-62(SEQ?ID?NO.：17)

4 D56-AA7(SEQ?ID?NO.：19)；D56-AE1(SEQ?ID?NO.：21)；185-BD3(SEQ?IDNO.：143)

5 D35-BB7(SEQ?ID?NO.：23)；D177-BA7(SEQ?ID?NO.：25)；D56A-AB6(SEQ?IDNO.：27)；D144-AE2(SEQ?ID?NO.：29)

6 D56-AG11(SEQ?ID?NO.：31)；D179-AA1(SEQ?ID?NO.：33)

7 D56-AC7(SEQ?ID?NO.：35)；D144-AD1(SEQ?ID?NO.：37)

8 D144-AB5(SEQ?ID?NO.：39)

9 D181-AB5(SEQ?ID?NO.：41)；D73-Ac9(SEQ?ID?NO.：43)

10 D56-AC12(SEQ?ID?NO.：45)

11 D58-AB9(SEQ?ID?NO.：47)；D56-AG9(SEQ?ID?NO.：49)；D56-AG6(SEQ?IDNO.：51)；D35-BG11(SEQ?ID?NO.：53)；D35-42(SEQ?ID?NO.：55)；D35-BA3(SEQID?NO.：57)；D34-57(SEQ?ID?NO.：59)；D34-52(SEQ?ID?NO.：61)；D34-25(SEQID?NO.：63)

12 D56-AD10(SEQ?ID?NO.：65)

13 56-AA11(SEQ?ID?NO.：67)

14 D177-BD5(SEQ?ID?NO.：69)；D177-BD7(SEQ?ID?NO.：83)

15 D56A-AG10(SEQ?ID?NO.：71)；D58-BC5(SEQ?ID?NO.：73)；D58-AD12(SEQ?IDNO.：75)

16 D56-AC11(SEQ?ID?NO.：77)；D35-39(SEQ?ID?NO.：79)；D58-BH4(SEQ?IDNO.：81)；D56-AD6(SEQ?ID?NO.：87)

17 D73A-AD6(SEQ?ID?NO.：89)；D70A-BA11(SEQ?ID?NO.：91)；D70A-BB5(SEQ?IDNO.：93)

18 D70A-AB5(SEQ?ID?NO.：95)；D70A-AA8(SEQ?ID?NO.：97)

19 D70A-AB8(SEQ?ID?NO.：99)；D70A-BH2(SEQ?ID?NO.：101)；D70A-AA4(SEQ?IDNO.：103)

20 D70A-BA1(SEQ?ID?NO.：105)；D70A-BA9(SEQ?ID?NO.：107)；D176-BG2(SEQ?IDNO.：141)

21 D70A-BD4(SEQ?ID?NO.：109)

22 D181-AC5(SEQ?ID?NO.：111)；D144-AH1(SEQ?ID?NO.：113)；D34-65(SEQ?IDNO.：115)

23 D35-BG2(SEQ?ID?NO.：117)

24 D73A-AH7(SEQ?ID?NO.：119)

25 D58-AA1(SEQ?ID?NO.：121)；D185-BC1(SEQ?ID?NO.：133)；D185-BG2(SEQ?IDNO.：135)

26 D73-AE10(SEQ?ID?NO.：123)

27 D56-AC12(SEQ?ID?NO.：125)

28 D177-BF7(SEQ?ID?NO.：127)；D185-BE1(SEQ?ID?NO.：137)；185-BD2(SEQ?IDNO.：139)

29 D73A-AG3(SEQ?ID?NO.：129)

30 D70A-AA12(SEQ?ID?NO.：131)；D176-BF2(SEQ?ID?NO.：85)

31 D176-BC3(SEQ?ID?NO.：145)

32 D176-BB3(SEQ?ID?NO.：147)

33 D186-AH4(SEQ?ID?NO.：5)

Embodiment 9: the amino acid sequence identity of separating acid fragment

Deduction is from the aminoacid sequence of embodiment 8 Cytochrome P450 fragment nucleotide sequences.Infer and distinguish corresponding to the amino acid that is right after behind GXRXCP (A/G) sequence motifs to C-terminal or terminator codon.On compared pieces sequence identity, to amino acid identity be 70% or higher sequence observe distinct packet.To amino acid identity be 80% or higher sequence observe preferred grouping, more preferably 90% amino acid identity or higher most preferably is grouped into 99% amino acid identity or higher.Group and group membership's corresponding aminoacid sequence is shown in Fig. 2.Find that some unique nucleic acid sequences and other fragment have amino acid identity completely, therefore only reported 1 member that same amino acid is arranged.

The amino acid identity of Table II group 19 on its nucleotide sequence basis corresponding to 3 not on the same group.Each group membership's aminoacid sequence and its identity are shown in Figure 77.The amino acid difference is by suitable mark.

At least 1 member of each amino acid identity group is selected for gene clone and uses the functional study of plant.In addition, handled by ethene to influence different group memberships or other biological variability by Northern and Southern analysis and evaluation is selected for gene clone and functional study.For assisting gene clone, expression study and the assessment of complete plant, peptide specific antibody prepare on sequence identity and diversity sequence basis.

Table II: tobacco P450 amino acid sequence identity group

The group fragment

1 D58-BG7(SEQ?ID?NO.：2)，D58-AB1(SEQ?ID?NO.：4)

2 D58-BE4(SEQ?ID?NO.：8)

3 D56-AH7(SEQ?ID?NO.：10)；D13a-5(SEQ?ID?NO.：12)

4 D56-AG10(SEQ?ID?NO.：14)；D34-62(SEQ?ID?NO.：18)

5 D56-AA7(SEQ?ID?NO.：20)；D56-AE1(SEQ?ID?NO.：22)；185-BD3(SEQ?IDNO.：144)

6 D35-BB7(SEQ?ID?NO.：24)；D177-BA7(SEQ?ID?NO.：26)；D56A-AB6(SEQ?IDNO.：28)；D144-AE2(SEQ?ID?NO.：30)

7 D56-AG11(SEQ?ID?NO.：32)；D179-AA1(SEQ?ID?NO.：34)

8 D56-AC7(SEQ?ID?NO.：36)；D144-AD1(SEQ?ID?NO.：38)

9 D144-AB5(SEQ?ID?NO.：40)

10 D181-AB5(SEQ?ID?NO.：42)；D73-Ac9(SEQ?ID?NO.：44)

11 D56-AC12(SEQ?ID?NO.：46)

12 D58-AB9(SEQ?ID?NO.：48)；D56-AG9(SEQ?ID?NO.：50)；D56-AG6(SEQ?IDNO.：52)；D35-BG11(SEQ?ID?NO.：54)；D35-42(SEQ?ID?NO.：56)；D35-BA3(SEQID?NO.：58)；D34-57(SEQ?ID?NO.：60)；D34-52(SEQ?ID?NO.：62)

13 D56AD10(SEQ?ID?NO.：66)

14 56-AA11(SEQ?ID?NO.：68)

15 D177-BD5(SEQ?ID?NO.：70)；D177-BD7(SEQ?ID?NO.：84)

16 D56A-AG10(SEQ?ID?NO.：72)；D58-BC5(SEQ?ID?NO.：74)；D58-AD12(SEQ?IDNO.：76)

17 D56-AC11(SEQ?ID?NO.：78)；D56-AD6(SEQ?ID?NO.：88)

18 D73A-AD6(SEQ?ID?NO.：90)；D70A-BB5(SEQ?ID?NO.：94)

19 D70A-AB5(SEQ?ID?NO.：96)；D70A-AB8(SEQ?ID?NO.：100)；D70A-BH2(SEQ?IDNO.：102)；D70A-AA4(SEQ?ID?NO.：104)；D70A-BA1(SEQ?ID?NO.：106)；D70A-BA9(SEQ?ID?NO.：108)；D176-BG2(SEQ?ID?NO.：142)

20 D70A-BD4(SEQ?ID?NO.：110)

21 D181-AC5(SEQ?ID?NO.：112)；D144-AH1(SEQ?ID?NO.：114)；D34-65(SEQ?IDNO.：116)

22 D35-BG2(SEQ?ID?NO.：118)

23 D73A-AH7(SEQ?ID?NO.：120)

24 D58-AA1(SEQ?ID?NO.：122)；D185-BC1(SEQ?ID?NO.：134)；D185-BG2(SEQ?IDNO.：136)

25 D73-AE10(SEQ?ID?NO.：124)

26 D56-AC12(SEQ?ID?NO.：126)

27 D177-BF7(SEQ?ID?NO.：128)；185-BD2(SEQ?ID?NO.：140)

28 D73A-AG3(SEQ?ID?NO.：130)

29 D70A-AA12(SEQ?ID?NO.：132)；D176-BF2(SEQ?ID?NO.：86)

30 D176-BC3(SEQ?ID?NO.：146)

31 D176-BB3(SEQ?ID?NO.：148)

32 D186-AH4(SEQ?ID?NO.：6)

Embodiment 10: clone's full-length cDNA P450 clone

The cDNA library handles from ethene by following that the total RNA of preparation makes up the leaf.At first, total RNA extracts from ethene and handles leaf, uses acid phenol and the chloroform extraction operation revised.Retouching operation is to use 1 gram tissue, and tissue is ground and extracts damping fluid (100mM Tris-HCl, pH8.5 at 5ml subsequently; 200mM NaCl; 10mMEDTA; 0.5%SDS) mesoscale eddies, damping fluid adds 5ml phenol (pH5.5) and 5ml chloroform.Centrifugal extraction sample is preserved supernatant.This extraction step repeats 2-3 time again and presents limpid up to supernatant.Add about 5ml chloroform to remove Determination of Trace Phenol.RNA is from mixing the supernatant partly precipitated, and this is by adding 3 times of volume ETOH and 1/10 volume 3M NaOAc (pH5.2) and preserving 1 hour at-20 ℃.After transferring to the Corex Glass Containers, it 4 ℃ with 9, centrifugal 45 minutes of 000RPM.Precipitation is washed with 70% ethanol, 4 ℃ with 9,000RPM rotated 5 minutes.After the precipitation drying, sedimentary RNA is dissolved in the water that 0.5ml does not have the RNA enzyme.The property quality and quantity of total RNA is respectively by sex change glutol and spectrophotometer analysis.

The total RNA of gained separates poly A+RNA by following operation, uses the operation of few d (T) Mierocrystalline cellulose (Invitrogen) and little centrifugal column spinner (Invitrogen).The total RNA of about 20mg carries out 2 purifying to obtain high quality poly A+RNA.Analyze poly A+RNA product, by the sex change glutol and subsequently the known full-length gene of RT-PCR to guarantee high quality mRNA.In addition, handle and change body leaf and ethene and handle the poly A+RNA that changes the body leaf and carry out Northern and analyze, use total length p450 as probe to handle non-transformation body leaf, zero hour ethene from ethene.The operation (KPL RNA detector Northern trace test kit) that method provides based on manufacturer's explanation, each sample uses 1.8 μ g poly A+RNA.The RNA that the contains glue transfer of spending the night uses 20X SSC as transfering buffering liquid.

Secondly, poly A+RNA to generate the cDNA library, uses cDNA synthetic agent box, ZAP-cDNA synthetic agent box, ZAP-cDNA Gigapack III gold clone test kit (Stratagene) as template.Method comprises manufacturer's operation of following detailed description.About 8 μ g poly A+RNA are used for the construction cDNA library.Analyze elementary library and show about 2.5 * 10 ⁶-1 * 10 ⁷Pfu.The quality background test in library is finished by the a-complementation, uses IPTG and X-gal, and the spot of wherein recombinating is to be higher than 100 times of expression of background response.

More show that the mean size that inserts cDNA is about 1.2kb in the quantitative analysis library by random PCR.Method is used two following step PCR method.For the 1st step, the design reverse primer is to obtain from the segmental preliminary sequence information of P450.The reverse primer of design and T3 (forward) primer are used for from the corresponding gene of cDNA amplified library.Agarose electrophoresis is carried out in PCR reaction, and corresponding high-molecular weight band is cut, purifying, clone and order-checking.In the 2nd step, clone to obtain total length p450 with the PCR that reverse primer (from P450 3 ' UTR design) is used from subsequently from the new primer of P450 5 ' UTR or start code district design as forward primer.

Total length p450 gene separates from the cDNA library that makes up by PCR method.2 PCR steps are used to clone full-length gene.In the 1st PCR step, non-special reverse primer (T3) and special forward primer (producing from the P450s downstream sequence) are used for from the 5 ' end of cDNA library clone P450s.The PCR fragment is separated, clone and order-checking, is used to design the forward primer of next step PCR.2 special primers are used for the 2nd PCR step clone total length p450 clone.Order-checking is subsequently cloned.

Expection those skilled in the art can make many modifications and variations according to the detailed description of foregoing invention in the practice invention.As a result, these modifications and variations are included in the following claim scope.

Claims (21)

1. isolated nucleic acid molecule, it is characterized in that the nucleotide sequence that described nucleic acid molecule comprises is selected from SEQ.ID.1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145,147 or 149.

2. isolated nucleic acid molecule as claimed in claim 1 is characterized in that described nucleic acid molecule comprises the fragment of cytochrome P450 gene.

3. an isolated nucleic acid molecule is characterized in that, the described nucleic acid molecule of described nucleic acid molecule and claim 1 has at least 75% identity.

4. an isolated nucleic acid molecule is characterized in that, the described nucleic acid molecule of described nucleic acid molecule and claim 1 has at least 91% identity.

5. an isolated nucleic acid molecule is characterized in that, the described nucleic acid molecule of described nucleic acid molecule and claim 1 has at least 99% identity.

6. transgenic plant is characterized in that, described transgenic plant comprise claim 1,2,3,4 or 5 described nucleic acid molecule.

7. transgenic plant as claimed in claim 1 is characterized in that described plant is a tobacco plant.

8. a method of making transgenic plant is characterized in that, said method comprising the steps of:

(i) promotor that function is arranged in claim 1,2,3,4 or 5 described nucleic acid molecule and the described plant can be operatively connected to produce plant conversion carrier; With

(ii) use the conversion carrier of the described plant of step (i) to transform described plant;

(iii) select by described conversion carrier plant transformed cell; And

(iv) from step described vegetable cell aftergrowth (iii).

9. method as claimed in claim 8 is characterized in that, described nucleic acid molecule is an antisense orientation.

10. method as claimed in claim 8 is characterized in that, described nucleic acid molecule is just direction.

11. method as claimed in claim 8 is characterized in that, described nucleic acid molecule is with the RNA interference radiating way.

12. method as claimed in claim 11 is characterized in that, described nucleic acid molecule is expressed as double stranded rna molecule.

13. method as claimed in claim 11 is characterized in that, described double stranded rna molecule length is about 15 to 25 Nucleotide.

14. method as claimed in claim 8 is characterized in that, described plant is a tobacco plant.

15. a selection contains the method for the plant of nucleic acid molecule, it is characterized in that, analyze the situation that exists of described plant nucleic acid sequence, wherein said nucleotide sequence is selected from SEQ.ID.1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145 or 147.

16. the method for selection plant as claimed in claim 15 is characterized in that described plant is by the DNA hybridization analysis.

17. the method for selection plant as claimed in claim 15 is characterized in that described plant is by the PCR check and analysis.

18. method as claimed in claim 16, it is characterized in that, described DNA hybridization comprises nucleic acid probe, and described nucleic acid probe is selected from SEQ.ID.1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131,133,135,137,139,141,143,145 or 147.

19. selection plant method as claimed in claim 15 is characterized in that described plant is transgenic plant.

20. selection plant method as claimed in claim 15 is characterized in that described plant is selected from mutagenized populations.

21. selection plant method as claimed in claim 15 is characterized in that described plant is selected from propagating population.

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