CN101130785A - Clone of rice WRKY gene relative to drought resistance and application thereof - Google Patents
Clone of rice WRKY gene relative to drought resistance and application thereof Download PDFInfo
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Abstract
The invention provides a WRKY protein related to the drought resistance from rice and the code gene. The transferred gene plant with the drought resistance improvement is acquired by leading the code gene or DNA sequence with isogeneic code and the same function in the plant structure, cell or organ and cultivating the plant cell, structure or organ into the plant. The experiment indicates that the gene transformed rice can improve the drought resistance of the rice, which doesn't affect the normal growth and the economic deseription of rice. The protein and the code gene has the important theory or actual meaning for researching the drought of the plant, improving the drought of the plant and changing the related deseription, which will show the important effect and has the wide application prospect in the improvement of the drought resistance gene engineering.
Description
Technical field
The present invention relates in the plant and coerce relevant transcription factor and encoding gene thereof and application, particularly relate to a WRKY transcription factor relevant that derives from paddy rice and encoding gene thereof and it is cultivating the application that improves in the drought tolerance plant with drought tolerance.
Background technology
Plant often suffers biology to coerce harm with abiotic stress in growth and development process, coerces in order to tackle these, and plant materials a series of coping mechanisms of having evolved out in the evolution of long period of time process are to resist various biologies and abiotic stress environment.In numerous coping mechanisms, the transcriptional regulatory of genetic expression is replied in the ambient signal irritant reaction process plant and is played important effect.Under the adverse environmental factor of environment-stress such as arid, high salt and low temperature, plant can be made corresponding adjustment on molecule, cell and integral level, with the injury that reduces environment to the full extent and caused and survived.A lot of genes are owing to be subjected to environment stress abduction delivering (Shinozaki, K.and Yamaguchi-Shinozaki, K. (1994) .Molecular responses to drought and cold stress.Curr.Opin.Biotechnol 7,161-167; Thomashow, M.F. (1999) .Plant cold acclimation:freezing tolerance genes and regulatory mechanisms.AnnuRev Plant Physiol Plant Mol Biol 50,571-599), the product of these genes not only can be participated in the stress response of plant directly, and can regulate other Expression of Related Genes or participate in signal transduction path, thereby plant is avoided or reduce injury, strengthen coercing the resistance of environment.In being subjected to environment stress abduction delivering process, transcription factor plays regulating and controlling effect to gene transcription and expression, is a kind of very important adjusting albumen in the plant materials.
Transcription factor is also referred to as trans-acting factor, thereby is can be conjugated protein with the DNA that also interaction activates or inhibition is transcribed of cis-acting elements specific combination in the eukaryotic gene promoter region.From structural analysis of protein, transcription factor is formed (Liu L by DNA land, transcription regulatory region (comprising active region or inhibitory area), oligomerization site (being used for identification) and nuclear localization signal usually, White M J, MacRae T H.Transcription factors and their genes in higher plantsfunctional domains, evolution and regulation.Eur J Biochem, 1999,262 (2), 247-257).Transcription factor interacts the transcript and expression of regulatory gene by the cis element effect in these functional areas and the gene promoter or with the functional area of other transcription factor.
In the whole genome of plant, the gene of the encoding transcription factor has accounted for greatly, such as, the gene of the encoding transcription factor has 1500 (Riechmann at least in the Arabidopis thaliana, J.L., Heard, J., Martin, G., Reuber, L., Jiang, C.-Z., Keddie, J., Adam, L., Pineda, O., Ratcliffe, O.J., Samaha, R.R., Creelman, R., Pilgrim, M., Broun, P., Zhang, J.-Z., Ghandehari, D., Sherman, B.K., and Yu, G.-L. (2000) .Arabidopsistranscription factors:genome-wide comparative analysis among eukaryotes.Science 290,2110), account for more than 5% of whole genome.These transcription factors belong to big gene family mostly, and the gene family that has can comprise many subtribes again, and some transcription factor family is that plant is peculiar.At present known in plant with coerce relevant transcription factor and mainly contain: AP2 (APETALA2)/EREBP (the ethylene responsive elementbinding protein) transcription factor family with AP2 structural domain, bZIP (basic region/leucinezipper motiftranscription factors) the class transcription factor that contains alkalescence zone and leucine zipper, the WRKY transcription factor family that contains conservative WRKY aminoacid sequence, the MYC family and MYB family of containing alkaline helix-loop-helix (bHLH) and leucine zipper with tryptophane bunch (Trp cluster).AP2 (APETALA2)/EREBP, bZIP, these four families of MYC, MYB all participate in regulating the environment stress reaction of plant to arid, high salt and low temperature etc.
WRKY albumen is a class transcription factor that only is present in the plant, contains 60 conservative amino acid whose WRKY structural domains, gains the name because of N end in this structural domain contains very conservative aminoacid sequence WRKYGQG.Also containing a kind of new zinc finger print body in the structural domain, is a class zinc finger protein.Nearly all WRKY albumen all has binding characteristic to W frame [(C/T) TGAC (C/T)], is that DNA is conjugated protein.
According to the feature of the phylogenetic relationship and the zinc fingers of WRKY structural domain, this family's factor is divided into eight subgroups of three big groups:
I group (comprising two subgroups of Ia and Ib) contains two WRKY structural domains usually, and the WRKY territory that is positioned at the C end has dna binding activity, and the WRKY territory of N end can not combine with DNA separately, and its function it be not immediately clear that the zinc fingers of this group is C2H2;
II group (comprising IIa, IIb, IIc and four subgroups of IId) contains a WRKY structural domain usually, and zinc fingers is identical with the I group, is C2H2;
III group (comprising two subgroups of IIIa and IIIb) also is to contain a WRKY structural domain, but its zinc fingers is C2HC.
WRKY albumen and the effect of W frame, all contain constant TGAC core sequence in the various W frames, this is essential (Yu D Q for the function of exercising the W frame and the combination of WRKY, Chen C H, Chen Z X.Evidence for an importantrole of WRKY DNA binding proteins in the regulation of NPR1 gene expression.Plant Cell, 2001,13 (7): 1527-1540; Sun C, Palmqvist S, Olsson H, et al.A novel WRKY transcriptionfactor, SUSIBA2, participates in sugar signaling in barley by binding to the sugar-reponsiveelements of the isol promoter.Plant Cell, 2003,15 (9): 2076-2092; Turck F, Zhou A, Somssich IE.Stimulus-dependent, promoter-specific binding of transcription factor WRKY1 toits nativepromoter and the defense-related gene PcPR1-1 inparsley.Plant Cell, 2004,16 (10): 2573-2585).The W frame has mediated the exciton inductive responsive transcription to pathogeny thing source, is present in the promotor of many and plant defense genes involved.Have the normal cluster of W frame of function to be present in the promoter sequence, a lot of WRKY genes self also all have W-box, hinting WRKY albumen may with other WRKY albumen or other class transcription factor actings in conjunction.
WRKY albumen is a distinctive class transcription factor family in the plant, in Arabidopis thaliana, there be 72-74 member (Eulgem T in WRKY family, Rushton P J, Robatzek S, et al.The WRKY superfamily of plant transcriptionfactors.Trends Plant Sci, 2000,5 (5): 199-206; Dong J X, Chen C H, Chen Z X.Expressionprofiles of the Arabidopsis WRKY gene suerfamily during plant defense response.Plant Mol Biol, 2003,51 (1): 21-37), a member surplus having 100 in the paddy rice, mainly be distributed in No. 1 and No. 5 karyomit(e) on (Wu K L, GuoZ J, Wang H H, et al.The WRKY family of transcription factors in rice and Arabidopsis andtheir origins.DNA Res, 2005,12 (1): 9-26; Zhang Y J, Wang L J.The WRKY transcription factorsuperfamily:its origin in eukaryotes and expansion in plants.BMC Evol Biol, 2005,5 (1): 1-12).
WRKY family gene function mainly be involved in plant disease resistance response, regulation and control damages induced gene, influence The Plant Senescence, involved in plant g and D process, resist environment stress etc.
WRKY gene family involved in plant defence disease resistance response.There is report to point out, after plant is infected by materials such as virus, bacterium, fungi excitons, the level of mRNA, protein and the dna binding activity thereof of WRKY gene all has raising (Wang Z P in the body, Yang P Z, Fan B F, et al.An oligo selectionprocedure for identification ofsequence-specific DNA-binding activities associated with plant defense.Plant J, 1998,16 (4): 515～522; Eulgem T, Rushton P J, Schmelzer E, et al.Early nuclear events in plant defensesignalling:rapid gene activation by WRKY transcription factors.EMBO J, 1999,18 (17): 4689～4699; Rushton P J, Torres J T, Pamiske M, et al.Interaction of elicitor-inducedDNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes.EMBO J, 1996,15 (20): 5690～5700; Fukuda Y.Interaction of tobacco nuclear protein with anelicitor-responsive element in the promoter of a basic class I chitinase gene.Plant Mol Biol, 1997,34 (1): 81～87).Arabidopis thaliana WRKY18 moderate horizontal expression can cause that the resistance that PR genetic expression reaches pseudomonas syringae (Pseudomonas syringae) strengthens (Chen C H, Chen Z X.Potentiation of developmentallyregulated plant defense response by AtWRKY18, a pathogen induced Arabidopsis transcriptionfactor.Plant Physiol, 2002,129 (2): 706～716).
The expression of WRKY gene regulating damage induced gene, AtWRKY6 is also prolonged in time and increases by abduction delivering after 1 hour in damage in the Arabidopis thaliana, in 6 hours, keep higher expression level (Robatzek S., and Somssich I.E., 2001, A new member of the A rabidopsis WRKY transcription factor family, AtWRKY6, isassociated with both senescence-and defense-related processes, Plant J., 28:123-133).The damage that is expressed in of tobacco leaf WIPK is handled after 3 minutes and promptly is significantly increased, and reaches maximum value in 5 minutes, falls back to initial value in 30 minutes.
The WRKY gene family is involved in plant development and substance metabolism process also, Arabidopis thaliana WRKY class transcription factor TTG2 gene is controlled three different form generating processes at least: trichome is grown, the generation of Weibull in generation of plant mucous and the inner seed coat in the outer kind skin, also may relevant (Johnson C S with root development, Kolevski B, Smyth D R.TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor.Plant Cell, 2002,14 (6): 1359～1375).OsWRKY71 is transcription inhibition factor (the Zhang Z L of GA signal transduction pathway in the gluten cell, Xie Z, Zou X, et al.A riceWRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleuronecells.Plant Physiol, 2004,134 (4): 1500～1513).
The aging course of WRKY class transcription factor involved in plant, Arabidopis thaliana WRKY53 gene participate in leaf senile and take place in early days.Robatzek etc. compare expression analysis to a plurality of Arabidopis thaliana WRKY genes, the result shows, the regulation and control of some WRKY class factor involved in plant aging course, they are that probe is studied the WRKY expression of gene in three different developmental phases blades of Arabidopis thaliana with 6 Arabidopis thaliana ESTs clones, found that, in spire and full ripe greenery, the several genes of other except that WRKY2,6 all have more weak expression; In old and feeble blade, WRKY4,6,7,11 has high level expression.They notice when the WRKY6 expression is analyzed in to plant different developmental phases different tissues simultaneously: this gene all has very weak expression in various tissues, but root with spend in relative stronger, in old and feeble blade, express the strongest, hint AtWRKY6 participates in aging course (the Hinderhofer K of blade, Zentgraf U.Identification of atranscription factor specifically expressed at the onset of leaf senescence.Planta, 2001,213 (3): 469～473; Robatzek S., and Somssich I.E., 2002, Targets of AtWRKY6regulation during plantsenescence and pathogen defense, Genes Dev., 16:1139-1149).
The WRKY family gene participates in the opposing environment stress, Chou Yuping etc. clone from low temperature (4 ℃) inductive rice leaf cDNA library and have obtained 13 WRKY genes, wherein there are 10 WRKY expression of gene to be subjected to the influence of NaCl, PEG, low temperature (4 ℃) and the high temperature abiotic stress factors such as (42 ℃), no matter and in adverse circumstance factor kind or on induction time, all there is very big-difference in their abduction delivering pattern.Wherein paddy rice WRKY30 gene is induced by PEG only, may set up and the important regulating and controlling effect is brought into play in aspect such as drought-resistant character formation at the signal transduction pathway that paddy rice is resisted the arid adverse circumstance factor.The molecular biology function of paddy rice WRKY16 gene may be mainly reflected in that adjusting and controlling rice opposing salt damage and the arid adverse circumstance factor coerce etc. that the biological character of aspect forms and corresponding signal transduction pathway is set up and gone up (Chou Yuping, chaste tree Shao Juan, pay hard, clone and expression pattern analysis thereof Deng .13 paddy rice WRKY gene. Science Bulletin, 2004,49 (18): 1860～1869).
In addition, plant hormone class signaling molecule has inducing action to the WRKY expression of gene, and plant materials can synthesize the phytoalexin PR albumen relevant with the course of disease when being subjected to the pathogeny thing and infecting, and makes plant obtain system's resistance (SAR).Studies show that Whitfield's ointment (SA), jasmonic (JA) and analogue thereof etc. are all closely related with SAR.WRKY albumen TDBA12 is induced at pathogen infection or SA processing back, TDBA12 can discern exciton response element (the Yang P Z in the tobacco chitinase promotor, Chen C H, Wang Z P, et al.A pathogen-and salicylic acid-induced WRKYDNA-binding activity recognizes the elicitor response element of tobacco class I chitinase genepromoter.Plant J, 1999,18 (2): 141～149).Four of discoveries such as Du can be by the inductive WRKY proteinoid identification of SA processing back by pathogen infection or SA inductive acceptor proteinoid kinases (RLK), these four kinase whose N ends have a signal sequence, an outer receptor domain of born of the same parents and a single membrane-spanning domain, the C end has a serine/threonine kinases territory, disclose effect (the Du L Q of WRKY proteinoid in disease-resistant signal transduction, Chen Z X.Identification of genes encodingreceptor-like protein kinases as possible targets of pathogen-and salicylic acid-induced WRKYDNA-binding proteins in Arabidopsis.Plant J, 2000,24 (6): 837～847).
The expression of WRKY gene pairs self has regulating effect, W-box in the AtWRKY18 gene promoter is a negative regulatory element, it may suppress the expression excessively of AtWRKY18 in the defense response, thereby the deleterious effect of this gene in growing process dropped to minimum, but Arabidopis thaliana WRKY transcription factor not all is to suppress son.AtWRKY6 plays restraining effect to the expression of himself and AtWRKY42, promoters driven gus reporter gene with AtWRKY6 detects less than the GUS activity in the CaMV35S:WRKY6 of overexpression protoplastis, has then found the expression of reporter gene in the WRKY of gene knockout mutant.
In sum, WRKY can sum up as shown in Figure 1 (high National Day, storage plant WRKY transcription factor family progress such as become a useful person, plant science circular, 2005,22 (1): 11～18) to the regulatory function of genetic transcription.
Summary of the invention
The purpose of this invention is to provide a kind of WRKY gene relevant with drought tolerance.
The WRKY gene relevant with drought tolerance provided by the present invention derives from paddy rice, coding following proteins (i) or (ii):
(i) has the aminoacid sequence of SEQ ID NO:1 in the sequence table;
(ii) in the aminoacid sequence that (i) limits through replacement, lack or add one to ten amino-acid residue and have regulation and control drought tolerance in plants sexual function by (i) deutero-protein.
SEQ ID NO:1 aminoacid sequence in the sequence table is made up of 674 amino-acid residues, wherein, is the DNA binding domains from aminoterminal 493-496 amino acids residue, will have the albumen called after OsWRKY30 of this amino acid residue sequence.One to ten amino-acid residue of described replacement, disappearance or interpolation can be the amino-acid residue in non-this structural domain, and its change can not exert an influence to this proteic function.
The present invention derives from the WRKY gene relevant with drought tolerance of paddy rice, can have following nucleotide sequence: the dna sequence dna of SEQ ID NO:2 in the sequence table.
SEQ ID NO:2 in the sequence table is by 2025 based compositions, its open reading frame is from 5 ' end 1-2O25 bit base, coding has the protein of the amino acid residue sequence of SEQ ID NO:1 in the sequence table, wherein, from 5 ' end 1477-1488 bit base coding DNA binding domains, the unnamed gene that will have this nucleotide sequence is OsWRKY30.
Contain expression carrier of the present invention, transgenic cell line and host bacterium all within protection scope of the present invention.
Arbitrary segmental primer is to also within protection scope of the present invention among the amplification OsWRKY30.
Another object of the present invention provides a kind of method that improves drought resistance in plants.
The method of raising drought resistance in plants provided by the present invention, be that the WRKY gene (having 90% above homology and the proteic dna sequence dna of coding identical function as OsWRKY30 or with this gene) that the present invention is relevant with drought tolerance imports plant tissue, cell or organ, drought resistance in plants obtains to improve.
In the method for above-mentioned raising drought resistance in plants, the paddy rice WRKY gene (OsWRKY30) that the present invention is relevant with drought tolerance both can be the cDNA sequence of described gene, also can be the genomic gene sequence of described gene; Having 90% above homology and coding identical function proteic dna sequence dna with described gene, is the cDNA of described gene or genomic gene sequence to be separated and/or modified and/or design with known method obtain.What it should be appreciated by those skilled in the art is; the minor alteration of Nucleotide identity may cause the reduction or the reinforcement of this gene usefulness in the specific gene sequence; and (for example in some application; antisense or suppress technology altogether) in, partial sequence plays a role equally effectively through regular meeting and full length sequence.The method that gene order changes or shortens, and the method for testing the validity of these genes that change all is well known to those skilled in the art.
Paddy rice WRKY gene (OsWRKY30) or its homologous sequence that the present invention is relevant with drought tolerance can import plant tissue, cell or organ by plant expression vector; The carrier that sets out that is used to make up described plant expression vector can be any one and can be used for the carrier etc. that agrobacterium tumefaciens or Agrobacterium rhizogenes transform the binary vector of plant or can be used for the plant micropellet bombardment, as pBin serial carrier (as pBin 19 etc.), pBI serial carrier (as pBI 101 etc.), Gateway serial carrier (as pH2GW7 etc.), pCAMBIA serial carrier (as pCAMBIA 3301 etc.), per8, pX6 or other plant expression vector of deriving, the described carrier that sets out also can be the carrier that can duplicate in prokaryotic organism, as pUC serial carrier or pBluescript serial carrier etc.
When using the present invention's paddy rice WRKY gene (OsWRKY30) relevant or its homologous sequence structure plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or induction type (ABA, arid, saline and alkaline or chemical induction etc.) promotor with drought tolerance; Described constructive expression's promotor can be cauliflower mosaic virus (CAMV) 35S promoter, corn Ubiquitin promotor or paddy rice actin1 promotor etc.; Described tissue specificity expression promoter can be root-specific expression promotor, blade specific is expressed promotor, dimension pipe specific expressing promoter, seed-specific expression promotor, flower specific expression promotor or pollen specific expression promotor, as 2S1 promotor (GenBank number: NM_118848.2, GI:30687489) and NapinA (GenBank number: M64633.1, GI:349405) promotor etc.; Described inducible promoter can be and is subjected to inductive promotors such as low temperature, arid, ABA, ethene, saline and alkaline or chemistry; Above-mentioned promotor can be used separately or be used in combination with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can comprise ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.
For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene (gus gene of luminophor as adding the coding that in plant, to express, the GFP gene, luciferase genes etc.), antibiotic marker thing [neomycin phosphotransferase (NPTII) gene with resistance, hygromix phosphotransferase (Hygromycin phosphotransferase) gene, gentamicin marker or kantlex marker etc.] or anti-chemical reagent marker gene (as anti-weedkiller gene) etc.Described host plant cell, tissue or the organ that contains neomycin phosphotransferase (NPTII) gene can be screened by kantlex or its substituted derivatives such as G418 etc., and the host plant cell, tissue or the organ that contain hygromix phosphotransferase (Hygromycinphosphotransferase) gene can be screened by Totomycin.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.After aforesaid method screens, also can adopt Southern, PCR or dot blot equimolecular detection means that transfer-gen plant is detected, whether transform goal gene to determine it.
Wherein, with the pH2GW7 (Gateway of Invitrogen company
TWThe vector carrier) be the carrier that sets out, the plant expression vector that contains the present invention's paddy rice WRKY gene relevant with drought tolerance of structure is pH2GW7-OsWRKY30.The plant expression vector that carries the present invention's paddy rice WRKY gene (OsWRKY30) relevant with drought tolerance or its homologous sequence can be by using protoplastis-chemical mediated method (Ca
2+, PEG), combination transformed plant cells, tissue or the organ of any or several method in sharp, the particle gun of Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversions, pollen tube importing, microinjection, electricity, conventional biological method such as agriculture bacillus mediated, and plant transformed cell, tissue or organ cultivated into plant; Described tissue and organ can comprise fruit pod, callus, stem apex, blade and the seed etc. of host plant.
In addition, after conversion being contained the present invention's paddy rice WRKY gene (OsWRKY30) relevant or carrying out succeeding transfer culture, can therefrom further filter out the transfer-gen plant of gene pure with transfer-gen plant that described gene has 90% above homology and a coding identical function proteic dna sequence dna with drought tolerance.In addition, also can expand this transfer-gen plant numerous, but the drought tolerance of render transgenic plant is further improved.The expansion of described transgenic plant is numerous to comprise vegetative propagation and/or seminal propagation.
Method of the present invention all is suitable for dicotyledons and monocotyledons, therefore, describedly both dicotyledonss such as tobacco, rape, cotton, soybean, willow, eucalyptus, potato or herbage can be derived from, also monocotyledonss such as paddy rice, corn, wheat, barley, jowar, millet or turfgrass can be derived from by plant transformed cell, tissue or organ.
The invention provides WRKY albumen and an encoding gene thereof relevant with drought tolerance.Experiment showed, gene transformation paddy rice of the present invention can be significantly improved the tolerance of paddy rice to drought stress, and normal growth and the economic characters of paddy rice are not significantly influenced.Albumen of the present invention and encoding gene thereof are for the drought tolerance in plants Study on Mechanism, and improve the drought tolerance of plant and the improvement of correlated character has important theory and practical significance, to in the drought-enduring genetically engineered improvement of plant (particularly cereal crop), play a significant role, have a extensive future.
Description of drawings
Fig. 1 is the regulating effect synoptic diagram of WRKY to genetic transcription.
Fig. 2 has illustrated OsWRKY30 to be subjected to ABA inductive expression pattern in paddy rice.
Fig. 3 is that PCR detects the electrophorogram of OsWRKY30 transgenosis T0 for the plant hygromycin phosphotransferase gene.
Fig. 4 is that OsWRKY30 transgenosis T1 behind drought stress is for the photo of plant and wild-type plant.
Fig. 5 is that OsWRKY30 transgenosis T1 is for the drought-enduring seedling per-cent of plant column diagram.
Fig. 6 is that OsWRKY30 transgenosis T2 behind drought stress is for the photo of plant and wild-type plant.
Fig. 7 is that OsWRKY30 transgenosis T2 is for the drought-enduring seedling per-cent of plant column diagram.
Embodiment
Below in conjunction with specific embodiment the present invention is described in further details.
Method therefor is ordinary method if no special instructions among the following embodiment, and concrete steps can be referring to " MolecularCloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning:ALaboratory Manual, 3
RdEdition, 2001, NY, Cold Spring Harbor).The primer and dna sequence dna are given birth to worker's biotechnology company limited by Shanghai and are synthesized.
ABA induces, spend 11 (Oryza SativaL.) in the ABA processing wild-type paddy rice with 100 μ M, drew materials at 0,1,2,6,12,24 hour respectively, the extracting RNA reverse transcription, with 5 '-ATGCCGCAAATGGACACCT-3 ' is forward primer, with 5 '-AACGCGAGGCTCCCGGATAG-3 ' is a reverse primer, source reference in the paddy rice Actin carries out PCR.1 hour as a result existing the expression, the increase expression amount increase with induction time reached maximum at 12 hours, later expression amount decline (see figure 2).
The acquisition of embodiment 2, the relevant OsWRKY30 gene of paddy rice with drought tolerance
1, the separation of OsWRKY30 and functional study
Retrieval ncbi database and document, sequence (the Gene bank Accession NO of acquisition WRKY30
AK065518), design forward primer: 5 '＞CACCACAAAATGGACGGGACCAACAACCATGGA＞3 ' and reverse primer 5 '＞CATCTGAGGATGCTGCTTTGGCAA＞3 ', spend the RNA of 11 paddy rice seedlings in the extraction PEG6000 processing, obtain cDNA by reverse transcription, the RT-PCR method is come out this gene clone, adopt TOPO clone's method to import pENTER-TOPO Vector, constitute pENTER-TOPO-OsWRKY30, pass through CaCl
2Method transformed into escherichia coli (E.coli) TOP10 bacterial strain, select positive bacterium colony and join 5ml and contain in the LB liquid nutrient medium of 50mg/L kantlex, cultivated 12-16 hour in 37 ℃, the shaking table of 220rpm, extract plasmid, PCR and enzyme cutting method carry out the sequencing analysis after identifying.
The acquisition of embodiment 3, the WRKY30 transgenic paddy rice relevant with drought tolerance
With agrobacterium-mediated transformation embodiment 2 is obtained with drought tolerance OsWRKY30 gene transformation paddy rice, concrete grammar is as follows:
1) transforms Agrobacterium
By the LR reaction recombinant plasmid pENTER-TOPO-OsWRKY30 that makes up among the embodiment 2 is imported the plant expression vector pH2GW7 (Gateway of Invitrogen company
TWThe vector carrier) in, the LR reaction system is 0.5 μ l LR enzyme, 1 μ l, 5 * damping fluid, and 2 μ l pENTER-TOPO-OsWRKY30,1.5 μ l pH2GW7, moisturizing 5 μ l, LR reaction conditions are that 25 ℃ of water-baths 1 were by 3 hours.Then by the heat shock method with above-mentioned recombinant vectors transformed into escherichia coli (E.coli) TOP10.With 5 '-CACCACAAAATGGACGGGACCAACAACCATGGA-3 ' is forward primer, 5 '-CATCTGAGGATGCTGCTTTGGCAA-3 ' is a reverse primer, the PCR screening positive clone, selecting positive single bacterium colony joins 5mL and contains in the LB liquid nutrient medium of spectinomycin of 50mg/L, at 37 ℃, 220rpm cultivated 12-16 hour down, extract plasmid, carry out enzyme with restriction enzyme EcorRV and cut evaluation, cut the enzyme that has obtained 1300bp and 800bp through enzyme and cut product, conform to expected results, qualification result shows the recombinant plant expression vector that has obtained to contain OsWRKY30, called after pH2GW7-OsWRKY30 transforms above-mentioned recombinant vectors pH2GW7-OsWRKY30 Agrobacterium AGL0 bacterial strain (Chinese Academy of Sciences's heredity is given) subsequently.Carry out the evaluation of positive reorganization Agrobacterium with forward primer 5 '-CACCACAAAATGGACGGGACCAACAACCATGGA-3 ' and reverse primer 5 '-TTACATCTGAGGATGCTGCTTTGGCAA-3 ' PCR, obtain the strain of AGL0 (pH2GW7-OsWRKY30) reorganization Agrobacterium.
2) infect the rice callus tissue
Single colony inoculation of the positive reorganization Agrobacterium that the picking step 1) obtains is in the 20mL YEB liquid nutrient medium that contains spectinomycin 50mg/L and Rifampin 50mg/L, under 28 ℃, 150rpm shaking culture 2-3 days, again in centrifugal 3 minutes of 4 ℃, 5000rpm, remove supernatant, bacterial sediment is resuspended in the AA substratum (Co (NO that contains the 0.1mmol/L Syringylethanone
3)
26H
2O 0.15mg/L, CaCl
2110mg/L, MgSO
4122mg/L, KI 3.75mg/L, NaH
2PO
4H
2O150mg/L, Na
2-EDTA 0.01mM, FeSO
47H
2O 139mg/L, KCl 2.95g/L, MnSO
44H
2O 84.5mg/L, ZnSO
47H
2O 6.25mg/L, H
3BO
35mg/L, Gly 37.5mg/L, CuSO
40.005mg/L, Na
2MoO
42H
2O1.25mg/L, vitamin VB
15mg/L, nicotinic acid 5mg/L, pyridoxine hydrochloride VB
65mg/L, creatine 0.1g/L, casein hydrolysate 0.3g/L, L-Gln 87.6mg/L, L-Asp 26.6mg/L, sucrose 20g/L) in, 28 ℃ of following lucifuge shaking culture 1-2 hour to OD
600=0.6-0.9.Select and spend in the paddy rice that method for plant tissue culture routinely obtains that 11 growth conditions is good, after the particulate state callus immerses and shakes 20 minutes in the reorganization Agrobacterium nutrient solution that above-mentioned conversion has OsWRKY30, left standstill 30 minutes, taking-up is blotted unnecessary bacterium liquid with aseptic filter paper, and callus is inoculated in N
6Be total to substratum (N
6Substratum+10g/L glucose+1mg/L Syringylethanone+2,4 dichlorophenoxyacetic acid (2,4-D) 2mg/L) is gone up and is cultivated, wherein N
6The prescription of minimum medium is: (NH
4)
2SO40.46g/L, KNO
32.83g/L, CaCl
20.2g/L, MgSO
40.092g/L, KH
2PO
40.4g/L, Na
2-EDTA0.15g/L, FeSO
47H
2O 0.11g/L, MnSO
44H
2O0.44g/L, ZnSO
47H
2O 0.17g/L, H
3BO
30.14g/L, CoCl
26H
2O 0.0005g/L, CuSO
45H
2O0.0005g/L, Na
2MoO
42H
2O 0.005g/L, vitamin VB
10.01g/L, nicotinic acid 0.001g/L, pyridoxine hydrochloride VB
60.001g/L, creatine 0.1g/L, casein hydrolysate 0.3g/L, L-Pro 0.5g/L, sucrose 30g/L, agar 10g/L, pH5.8.After 2-3 days, callus is put into wide-necked bottle, with aseptic water washing 3-5 time, shake for several times at every turn, in water, lose thread thalline, in the sterilized water that contains the 500mg/L cephamycin, soaked 30-60 minute then, use aseptic water washing 1 time at last again, place airing on the aseptic filter paper, change N at last over to
6Cultivate altogether in the substratum altogether.
3) acquisition of positive transgenic paddy rice
Rice callus after will infecting is organized in N
6After cultivating 3-5 days altogether in the substratum altogether, go to the N that contains 30mg/L Totomycin and the full mycin of 400mg/L head
6Solid medium (N
6Macroelement+B
5Trace element+B
5Organic composition+300mg/L caseinhydrolysate+500mg/L proline(Pro)+30g/L sucrose+7-8g/L agar, pH 5.8) go up 3 weeks of screening, change the N that contains 50mg/L Totomycin and the full mycin of 200mg/L head again over to
64 weeks of screening change resistant calli over to pre-differentiation substratum (N subsequently on the solid medium
6Substratum+1mg/L naa+2mg/L 6-benzylaminopurine+5mg/L dormin), in 3 weeks of illumination cultivation, change division culture medium (N again over to
6Substratum+1mg/L naa+2mg/L 6-benzylaminopurine) breaks up on, move in the greenhouse regeneration plant that grows carried out root culture on strong seedling culture base (1/2MS inorganic salt+0.5mg/L naa+0.25mg/L paclobutrazol) after, at 28 ℃, 15 hours/day illumination is cultivated after 1 month down and is got plant leaf, extract total DNA according to a conventional method, with 5 '-ACT CAC CGC GAC GTC TGT-3 ' and 5 '-TTC CTT TGC CCTCGG ACG-3 ' is forward and reverse primer, the pcr amplification hygromycin phosphotransferase gene, obtain the positive transfer-gen plant of about 1100bp size dna fragmentation through amplification, detected result as shown in Figure 3,1-8 is a transgenic seedling, M is DNAMarker, and WT is a wild-type.Show with aforesaid method and obtained to transform the transgenic paddy rice that OsWRKY30 is arranged.
T0 is for the OsWRKY30 transgenic paddy rice seed for results, choose 10 different transgenic lines, each strain system chooses 100 full seeds and soaks seed, behind the germination and emergence, Totomycin with 25mg/L screened 5 days, choose strain system, obtain 7 through screening and have the positive transgenic lines that unit point inserts with unit point insertion.
To go into water content in the nutrition soil of 55%-65% through the positive transgenic paddy rice seed after the hygromycin selection with unit point insertion, do not rewater from this very day from handling, until the withered jaundice of wild-type CK plant leaf, wilting, death, root system attenuates, begin to send out brown, the OsWRKY30 transfer-gen plant is normal growth still, and seedling leaf still is green, and the root system extent of damage is less.OsWRKY30 T1 for the seedling of transgenic line after arid is handled as shown in Figure 4, three young plants on the left side are the wild-type plant in every photo, three young plants on the right are that OsWRKY30 transgenosis T1 is for plant.Remove stress conditions, rehydration restore normal growth seedling, and statistics survives the seedling number after 2 weeks, calculates the drought-enduring seedling per-cent of each strain system, is contrast (CK) with the wild-type plant, and the drought tolerance of OsWRKY30 transgenic paddy rice seedling obviously improves (see figure 5).
T1 is for the OsWRKY30 transgenic paddy rice seed for results, T1 is carried out T2 for the drought-enduring evaluation of plant for 5 different transgenic lines with unit point insertion that carry out after arid is screened, each strain system chooses 3 different T2 and screens for plant, each plant is chosen 100 full seeds and soaks seed, behind the germination and emergence, Totomycin with 25mg/L screened 5 days, chose the homozygote plant and carried out drought-enduring screening.Drought-enduring screening conditions and T1 for the time the drought-enduring screening conditions of carrying out the same, to go into water content through the positive transgenic paddy rice seed after the hygromycin selection in the nutrition soil of 55%-65%, do not rewater from this very day from handling, till contrasting the not withered death of transgenic paddy rice.OsWRKY30T2 for the seedling of transgenic line after arid is handled as shown in Figure 6, three young plants on the left side are the wild-type plant in every photo, three young plants on the right are that OsWRKY30 transgenosis T2 is for plant.Remove stress conditions, rehydration restore normal growth seedling, and 2 week back statistics surviving the seedling number, and calculating each strain is the drought-enduring seedling per-cent of 3 plant, and the drought tolerance of OsWRKY30 transfer-gen plant is than the (see figure 7) that increases of the drought tolerance of transfer-gen plant (CK) not.
Sequence table (SEQUENCE LISTING)
＜110〉Weimingkaituo Agro-Biological Technology Co., Ltd., Beijing
＜120〉clone and application with drought-enduring relevant paddy rice WRKY gene
<130>JSP070141
<160>2
<170>PatentIn?version?3.1
<210>1
<211>674
<212>PRT
＜213〉spend 11 (Oryza Sativa L.) in the paddy rice
<400>1
Met?Asp?Gly?Thr?Asn?Asn?His?Gly?Ala?Leu?Met?Asp?Asp?Trp?Met?Leu
1 5 10 15
Pro?Ser?Pro?Ser?Pro?Arg?Thr?Leu?Met?Ser?Ser?Phe?Leu?Asn?Glu?Glu
20 25 30
Phe?Ser?Ser?Gly?Pro?Phe?Ser?Asp?Ile?Phe?Cys?Asp?Asn?Gly?Ser?Asn
35 40 45
Lys?His?Gln?Asp?Gly?Leu?Gly?Lys?Ser?Lys?Ala?Phe?Ile?Asp?Ser?Ser
50 55 60
Arg?Glu?Glu?Thr?Ala?Gln?Leu?Ala?Lys?Lys?Phe?Glu?Ser?Asn?Leu?Phe
65 70 75 80
Gly?Ala?Asn?Gln?Lys?Ser?Ser?Ser?Asn?Gly?Cys?Leu?Ser?Glu?Arg?Met
85 90 95
Ala?Ala?Arg?Thr?Gly?Phe?Gly?Val?Leu?Lys?Ile?Asp?Thr?Ser?Arg?Val
100 105 110
Gly?Tyr?Ser?Thr?Pro?Ile?Arg?Ser?Pro?Val?Thr?Ile?Pro?Pro?Gly?Val
115 120 125
Ser?Pro?Arg?Glu?Leu?Leu?Glu?Ser?Pro?Val?Phe?Leu?Pro?Asn?Ala?Ile
130 135 140
Ala?Gln?Pro?Ser?Pro?Thr?Thr?Gly?Lys?Leu?Pro?Phe?Leu?Met?His?Ser
145 150 155 160
Asn?Val?Lys?Pro?Ser?Ile?Pro?Lys?Lys?Thr?Glu?Asp?Glu?Thr?Arg?His
165 170 175
Asp?Arg?Val?Phe?Phe?Phe?Gln?Pro?Ile?Leu?Gly?Ser?Lys?Pro?Pro?Thr
180 185 190
Cys?Pro?Val?Ala?Glu?Lys?Gly?Phe?Ser?Val?Asn?His?Gln?Asn?Gln?Pro
195 200 205
Ser?Val?Thr?Asp?Asn?His?Gln?Glu?Leu?Ser?Leu?Gln?Ser?Ser?Ser?Thr
210 215 220
Ala?Ala?Lys?Asp?Phe?Thr?Ser?Ala?Thr?Ile?Val?Lys?Pro?Lys?Thr?Ser
225 230 235 240
Asp?Ser?Met?Leu?Asp?Asn?Asp?Asp?His?Pro?Ser?Pro?Ala?Asn?Asp?Gln
245 250 255
Glu?Glu?Asn?Ala?Thr?Asn?Lys?Asn?Glu?Glu?Tyr?Ser?Ser?Asp?Leu?Ile
260 265 270
Ile?Thr?Pro?Ala?Glu?Asp?Gly?Tyr?Asn?Trp?Arg?Lys?Tyr?Gly?Gln?Lys
275 280 285
Gln?Val?Lys?Asn?Ser?Glu?His?Pro?Arg?Ser?Tyr?Tyr?Lys?Cys?Thr?Phe
290 295 300
Thr?Asn?Cys?Ala?Val?Lys?Lys?Val?Glu?Arg?Ser?Gln?Asp?Gly?Gln?Ile
305 310 315 320
Thr?Glu?Ile?Val?Tyr?Lys?Gly?Ser?His?Asn?His?Pro?Leu?Pro?Pro?Ser
325 330 335
Asn?Arg?Arg?Pro?Asn?Val?Pro?Phe?Ser?His?Phe?Asn?Asp?Leu?Arg?Asp
340 345 350
Asp?His?Ser?Glu?Lys?Phe?Gly?Ser?Lys?Ser?Gly?Gln?Ala?Thr?Ala?Thr
355 360 365
Ser?Trp?Glu?Asn?Ala?Ala?Asn?Gly?His?Leu?Gln?Asp?Val?Gly?Ser?Glu
370 375 380
Val?Leu?Thr?Lys?Leu?Ser?Ala?Ser?Leu?Thr?Thr?Thr?Glu?His?Ala?Glu
385 390 395 400
Lys?Ser?Val?Met?Asp?Lys?Gln?Glu?Ala?Val?Asp?Ile?Ser?Ser?Thr?Leu
405 410 415
Ser?Asn?Glu?Glu?Asp?Asp?Arg?Val?Thr?His?Arg?Ala?Pro?Leu?Ser?Leu
420 425 430
Gly?Phe?Asp?Ala?Asn?Asp?Asp?Tyr?Val?Glu?His?Lys?Arg?Arg?Lys?Met
435 440 445
Asp?Val?Tyr?Ala?Ala?Thr?Ser?Thr?Ser?Thr?Asn?Ala?Ile?Asp?Ile?Gly
450 455 460
Ala?Val?Ala?Ser?Arg?Ala?Ile?Arg?Glu?Pro?Arg?Val?Val?Val?Gln?Thr
465 470 475 480
Thr?Ser?Glu?Val?Asp?Ile?Leu?Asp?Asp?Gly?Tyr?Arg?Trp?Arg?Lys?Tyr
485 490 495
Gly?Gln?Lys?Val?Val?Lys?Gly?Asn?Pro?Asn?Pro?Arg?Ser?Tyr?Tyr?Lys
500 505 510
Cys?Thr?His?Pro?Gly?Cys?Ser?Val?Arg?Lys?His?Val?Glu?Arg?Ser?Ser
515 520 525
His?Asp?Leu?Lys?Ser?Val?Ile?Thr?Thr?Tyr?Glu?Gly?Lys?His?Asn?His
530 535 540
Glu?Val?Pro?Ala?Ala?Arg?Asn?Ser?Gly?His?Pro?Ser?Ser?Gly?Ser?Ala
545 550 555 560
Ala?Ala?Pro?Gln?Ala?Thr?Asn?Gly?Leu?Leu?His?Arg?Arg?Pro?Glu?Pro
565 570 575
Ala?Gln?Gly?Gly?Gly?Gly?Gly?Ser?Leu?Ala?Gln?Phe?Gly?Tyr?Gly?Ser
580 585 590
Ala?Gly?His?Arg?Pro?Ala?Glu?Gln?Phe?Gly?Ala?Ala?Ala?Ala?Gly?Phe
595 600 605
Ser?Phe?Gly?Met?Leu?Pro?Arg?Ser?Ile?Ala?Thr?Pro?Ala?Pro?Ser?Pro
610 615 620
Ala?Ile?Ala?Val?Pro?Ala?Met?Gln?Gly?Tyr?Pro?Gly?Leu?Val?Leu?Pro
625 630 635 640
Arg?Gly?Glu?Met?Lys?Val?Asn?Leu?Leu?Pro?Gln?Ser?Gly?Asn?Ala?Gly
645 650 655
Ala?Ala?Ala?Ser?Gln?Gln?Leu?Met?Gly?Arg?Leu?Pro?Lys?Gln?His?Pro
660 665 670
Gln?Met
<210>2
<211>2025
<212>DNA
＜213〉spend 11 (Oryza Sativa L.) in the paddy rice
<400>2
atggacggga?ccaacaacca?tggagcactg?atggacgatt?ggatgcttcc?ctcacccagt 60
ccaagaacac?tcatgtcgag?tttcttgaac?gaagaattca?gctccggtcc?cttttcagac 120
attttctgtg?ataatggcag?taacaaacat?caggatggac?ttgggaagag?caaagctttc 180
atcgattcaa?gccgggaaga?aactgctcag?ctagcaaaaa?agtttgaatc?aaaccttttt 240
ggtgccaacc?agaaatcaag?ctcaaatggc?tgtctgtcag?agaggatggc?tgcaaggaca 300
ggttttggtg?tcctgaaaat?tgatacatct?cgtgtcggtt?attctacacc?gattcggtct 360
ccggtgacga?tcccgcccgg?tgtgagtcca?agggaacttc?ttgagtcgcc?ggtttttctt 420
ccgaacgcca?ttgcacaacc?ttctcctacc?actggcaaac?tgccattttt?gatgcatagt 480
aatgttaaac?catcgatccc?taaaaaaact?gaagatgaaa?cacgccatga?tcgtgtattc 540
ttctttcaac?ccattttggg?atctaagcca?ccaacttgtc?cagttgcaga?gaagggtttc 600
agtgttaatc?atcaaaacca?gccttcagtg?acggataatc?accaggagct?cagtcttcag 660
tctagctcaa?ctgcagccaa?ggatttcact?tcagcaacta?ttgttaaacc?taagacatct 720
gattccatgt?tagacaatga?tgatcaccct?tcccctgcaa?atgatcaaga?agagaatgca 780
acaaacaaaa?atgaagagta?ttcttcagac?ctgatcatta?cccctgctga?ggatggatat 840
aactggagga?aatatggaca?gaagcaagtt?aagaacagtg?agcatcccag?aagctactac 900
aaatgcactt?tcacgaattg?cgctgtcaag?aaggtggagc?gttctcaaga?tggccaaata 960
acagagatag?tctacaaagg?ttctcacaat?caccctttgc?cgccttccaa?ccgccgacca 1020
aatgttcctt?tctcacactt?caatgatctg?agagatgatc?actctgagaa?atttggttcc 1080
aagtctggtc?aggccacagc?aacttcatgg?gagaatgccg?caaatggaca?cctccaagat 1140
gtcggtagtg?aagttctgac?aaaactgtct?gcttctctta?cgacaacaga?acatgctgaa 1200
aaatctgtta?tggacaaaca?agaagctgtg?gatatctcat?cgacgctctc?caatgaagag 1260
gatgataggg?taacgcatcg?tgccccgctt?tctctgggct?ttgatgcgaa?cgatgactat 1320
gttgaacaca?agagaagaaa?gatggatgtt?tatgccgcta?ctagcactag?caccaacgcc 1380
atcgacatag?gagctgtggc?gtcaagagct?atccgggagc?ctcgcgttgt?tgttcagacc 1440
acaagtgagg?ttgacatcct?tgatgatggt?taccgttggc?gcaagtatgg?gcagaaagtt 1500
gtcaaaggaa?acccaaatcc?aaggagctac?tacaagtgca?ctcatccggg?ttgctcggtg 1560
cgcaagcatg?tggagcgatc?atcgcatgat?ctgaaatccg?tcatcacgac?gtatgaagga 1620
aagcacaacc?atgaagttcc?agctgccagg?aacagtggcc?acccaagctc?aggctcagcc 1680
gctgcaccac?aggctaccaa?tggtcttctt?caccggagac?ctgaaccggc?acaaggtggt 1740
ggtggtggta?gccttgctca?gtttggctat?ggctcagctg?gtcacagacc?agcagagcag 1800
tttggtgcag?cagcagctgg?tttctccttt?ggaatgctgc?ctcgtagcat?tgcaactccg 1860
gcgccgtctc?cggcgatcgc?cgtgccggcg?atgcaggggt?acccagggct?tgtgctgccg 1920
agaggtgaga?tgaaggtgaa?cttgctgcca?cagtctggga?atgctggtgc?agcagctagc 1980
cagcagctga?tgggcaggtt?gccaaagcag?catcctcaga?tgtaa 2025
Claims (10)
1. method that improves drought resistance in plants, be WRKY gene transfered plant tissue, cell or the organ that drought tolerance is relevant, to be cultivated into plant by plant transformed cell, tissue or organ again, obtain the transgenic plant that drought tolerance improves, the following protein of WRKY genes encoding (i) that described drought tolerance is relevant or (ii):
(i) has the aminoacid sequence of SEQ ID NO:1 in the sequence table;
(ii) in the aminoacid sequence that (i) limits through replacement, lack or add one to ten amino-acid residue and have improve the drought tolerance in plants sexual function by (i) deutero-protein.
2. the method for raising drought resistance in plants according to claim 1 is characterized in that: the WRKY gene that described drought tolerance is relevant has the nucleotide sequence of SEQ ID NO:2 in the sequence table.
3. the method for raising drought resistance in plants according to claim 1 and 2 is characterized in that: the WRKY gene that described drought tolerance is relevant imports plant tissue, cell or organ by plant expression vector.
4. the method for raising drought resistance in plants according to claim 3, it is characterized in that: the carrier that sets out that is used to make up described plant expression vector is a kind ofly to can be used for the carrier that agrobacterium tumefaciens or Agrobacterium rhizogenes transform the binary vector of plant or can be used for the plant micropellet bombardment, or the carrier that can duplicate in prokaryotic organism.
5. the method for raising drought resistance in plants according to claim 4 is characterized in that: described to can be used for the carrier that agrobacterium tumefaciens or Agrobacterium rhizogenes transform the binary vector of plant or can be used for the plant micropellet bombardment be pBin serial carrier, pBI serial carrier, Gateway serial carrier, pCAMBIA serial carrier, per8 or pX6; The described carrier that can duplicate in prokaryotic organism is pUC serial carrier or pBluescript serial carrier.
6. the method for raising drought resistance in plants according to claim 5 is characterized in that: the described carrier that sets out is pH2GW7.
7. the method for raising drought resistance in plants according to claim 3, it is characterized in that: during with the relevant gene constructed plant expression vector of WRKY of described drought tolerance, before its transcription initiation Nucleotide, add a kind of enhancement type, composing type, organizing specific type or inducible promoter.
8. the method for raising drought resistance in plants according to claim 3 is characterized in that: add translational enhancer and/or transcriptional enhancer during with the relevant gene constructed plant expression vector of WRKY of described drought tolerance.
9. the method for raising drought resistance in plants according to claim 3, it is characterized in that: in described plant expression vector, add the coding that in plant, to express and can produce the enzyme of colour-change or the gene of luminophor, perhaps add antibiotic marker thing or anti-chemical reagent marker gene with resistance.
10. the method for raising drought resistance in plants according to claim 1, it is characterized in that: described vegetable cell, tissue or organ origin are in dicotyledons or monocotyledons, and described dicotyledons comprises tobacco, rape, cotton, soybean, willow, eucalyptus, potato and herbage; Described monocotyledons comprises paddy rice, corn, wheat, barley, jowar, millet or turfgrass.
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| CN2007101196828A CN101130785B (en) | 2007-07-30 | 2007-07-30 | Clone of rice WRKY gene relative to drought resistance and application thereof |
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| CN2007101196828A CN101130785B (en) | 2007-07-30 | 2007-07-30 | Clone of rice WRKY gene relative to drought resistance and application thereof |
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