CN103614385A - Application of gene KT525 to improvement of stress tolerance of plants - Google Patents

Abstract

The invention discloses a gene and an application thereof, wherein the gene is from rice and is relevant to stress tolerance. Experimental results show that the tolerance of rice to drought and salt stress can be remarkably improved through transforming the gene to the rice. A protein and an encoding gene thereof have important theoretical and practical significance for the research on a plant stress tolerance mechanism and improvement on the stress tolerance and correlated characters of the plants, can play an important role in improving the stress-resistance gene engineering of the plants (particularly cereal crops) and are wide in application prospect.

Description

A gene KT525 is in the application improving on plant stress tolerance

Technical field

The present invention relates to gene and its application that plant stress tolerance is relevant, particularly derive from paddy rice to degeneration-resistant relevant gene KT525 in the application improving on plant stress tolerance.

Background technology

Paddy rice, corn and wheat are the important food crop of China, and the output of three large crops, quality are all most important for grain-production and the grain security of China.The abiotic stresses such as arid, saline and alkaline, high temperature and freeze injury can directly affect normal growth and the output of food crop.Utilize Modern Agricultural Biotechnology, as transgenic technology, cultivate and there is resistance of reverse and extensive adaptive new crop varieties, can make farm crop under adverse environmental factor, keep stablizing high yield.Along with going deep into of transgenic research, successively separating clone some and degeneration-resistant relevant gene, comprise key controlling gene, osmoregulation albumen, small-molecule substance in metabolic process, also have to participate in the transcription factor of the various Stress response approach of regulation and control etc.

Transcription factor is the key gene of regulate gene expression, and growing of crop played to important regulating effect.For many years, the clone of transcription factor and functional study are one of focuses of scientific research always, a large amount of transcription factor that scientists has been utilized different study route isolation identification, also some regulatory factors relevant to economical character have therefrom been found, for the character improvements of farm crop provides important gene resource.After Sequencing of Rice Genome work completes, many databases are analyzed rice genome.It is predicted, in long-grained nonglutinous rice and japonica rice, comprise respectively 2,025 and 2,384 transcription factors, belong to 63Ge transcription factor gene family, as WRKY(111/113, long-grained nonglutinous rice/japonica rice), bZIP(88/109), AP2/EREBP(174/182), AUX/IAA(30/46), MYB(136/138), HB(84/103) etc.According to bibliographical information in the past and the functional annotation of database, here both comprised the peculiar transcription factor family of plant, as WRKY, also comprise and closely-related some other transcription factor families such as growth and development of plants, resistance, as bZIP, AP2/EREBP, MYB etc.Utilize these databases, in conjunction with gene expression informations such as gene chip hybridization, est sequences, from genomic level prediction and separating rice transcription factor full-length cDNA, and utilize the efficient rice conversion system of having set up to make its overexpression in paddy rice, it is carried out to mass-producing functional verification, further by the phenotype variation of research transgenic paddy rice and the change of anti-adversity etc., infer the function of transcription factor; On the basis of the work such as a large amount of repeated Plant Transformation, phenotype analytical, Function Identification, searching has the important regulatory factor of plant of practical value in improvement farm crop, and be applied to the character improvement of crop, practical problems in effective solution agriculture production, has important theory significance and using value.

Summary of the invention

The object of this invention is to provide one section of nucleotide sequence relevant to rice stress-tolerance, called after KT525 gene, for improving the resistance of reverse energy of plant.

The KT525 gene relevant to resistance of reverse that provide of invention, derive from Oryza paddy rice ( oryza satival.), coding has the protein of aminoacid sequence shown in the SEQ ID NO:1 in sequence table.

SEQ ID NO:1 in sequence table is comprised of 263 amino-acid residues, is albumen KT525.In the present invention, the encoding gene of KT525 both can be the cDNA sequence of described gene, also can be the genomic dna sequence of described gene, or had 90% above homology and the DNA sequence dna of the identical function albumen of encoding with described gene.The encoding gene with aminoacid sequence shown in SEQ ID NO:1, can have the nucleotide sequence of SEQ ID NO:2 in sequence table.

The expression vector that contains gene of the present invention, transgenic cell line and Host Strains all belong to protection scope of the present invention.

The primer pair of the arbitrary fragment of amplification KT525 is also within protection scope of the present invention.

Another object of the present invention is to provide a kind of method that improves plant stress tolerance.The method of raising plant stress tolerance provided by the present invention is that plant stress tolerance obtains and improves by code book invention KT525 gene transfered plant tissue, cell or the organ relevant to resistance of reverse.

In the method for above-mentioned raising plant stress tolerance, the KT525 gene that in the present invention, paddy rice is relevant to resistance of reverse both can be the cDNA sequence of described gene, also can be the genomic gene sequence of described gene; Having the DNA sequence dna of 90% above homology and coding identical function albumen with described gene, is the cDNA of described gene or genomic gene sequence to be carried out to separation and/or modification and/or design by known method obtain.What it should be appreciated by those skilled in the art is; in specific gene sequence, the minor alteration of Nucleotide identity may cause reduction or the reinforcement of this gene usefulness; and in some application (for example; antisense or co-suppression technology) in, the frequent meeting of partial sequence and full length sequence play a role equally effectively.The method that gene order changes or shortens, and the method for testing the validity of these genes that change is all well known to those skilled in the art.

Paddy rice of the present invention KT525 gene relevant to resistance of reverse or its homologous sequence can import plant tissue, cell or organ by plant expression vector; For the carrier that sets out that builds described plant expression vector, can be the carrier etc. that any one can be used for the binary vector of agrobacterium tumefaciens or Agrobacterium rhizogenes conversion of plant or can be used for plant micropellet bombardment, as pBin serial carrier (as pBin 19 etc.), pBI serial carrier (as pBI 101 etc.), Gateway ^tWserial carrier (as pH2GW7 etc.), pCAMBIA serial carrier (as pCAMBIA 3301 etc.), per8, pX6 or other derivative plant expression vector, the described carrier that sets out also can be the carrier that can copy in prokaryotic organism, as pENTER-TOPO, pUC serial carrier or pBluescript serial carrier etc.

While using KT525 gene that in the present invention, paddy rice is relevant to resistance of reverse 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.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 (No. GenBank: NM_118848.2, GI:30687489) and No. NapinA(GenBank: M64633.1, GI:349405) promotor etc.; Described inducible promoter can be the promotor that is subject to low temperature, arid, ABA, ethene, the induction such as saline and alkaline or chemical.Above-mentioned promotor can be used alone or is combined with other plant promoter.In addition, while using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer and/or transcriptional enhancer, these enhanser regions can be 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 synthesize.Translation initiation region can be from transcription initiation region or structure gene.

For the ease of transgenic plant cells or plant are identified and are screened, can process plant expression vector used, the coding that can express in plant as added can produce the enzyme of colour-change or the gene (gus gene of luminophor, 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 organ containing neomycin phosphotransferase (NPTII) gene can be screened as G418 etc. by kantlex or its substituted derivatives, containing host plant cell, tissue or the organ of hygromix phosphotransferase (Hygromycin phosphotransferase) 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 screening, aforesaid method also can adopt Southern, PCR or dot blot equimolecular detection means to detect transfer-gen plant, to determine whether it has transformed goal gene.

Wherein, the present invention with pCAMBIA1300 for the carrier that sets out, the plant expression vector called after pCactF-KT525 that contains the KT525 gene that paddy rice of the present invention is relevant to resistance of reverse of structure.The plant expression vector that carries KT525 gene that paddy rice of the present invention is relevant to resistance of reverse or its homologous sequence can be by being used protoplastis-chemical mediated method (Ca ²⁺, PEG), Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, pollen tube importing, microinjection, electricity swash, combination transformed plant cells, tissue or the organ of any one or more method in particle gun, the conventional biological method such as agriculture bacillus mediated, and the vegetable cell of conversion, 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,, by conversion being had KT525 gene that paddy rice of the present invention is relevant to resistance of reverse or the transfer-gen plant of its homologous sequence carry out after succeeding transfer culture, can therefrom further filter out the transfer-gen plant of gene pure.In addition, also can expand this transfer-gen plant numerously, resistance of reverse that can render transgenic plant is further improved.The expansions of described transgenic plant is numerous comprises vegetative propagation and/or seminal propagation.

Method of the present invention is all applicable to dicotyledons and monocotyledons, therefore, the described vegetable cell being converted, tissue or organ both can derive from the dicotyledonss such as tobacco, rape, cotton, soybean, willow, eucalyptus, potato or herbage, also can derive from the monocotyledonss such as paddy rice, corn, wheat, barley, jowar, millet or turfgrass.

The invention provides a gene KT525 relevant to resistance of reverse.Experiment showed, gene transformation paddy rice of the present invention can be improved to the tolerance of paddy rice to arid, salt stress.Albumen of the present invention and encoding gene thereof are for the resistance to contrary machine-processed research of plant, and improve the resistance of reverse of plant and the improvement of correlated character has important theory and practical significance, by playing a significant role in the resistance to contrary genetically engineered improvement of plant (particularly cereal crop), have a extensive future.

Below in conjunction with specific embodiment, the present invention is described in further details.

Accompanying drawing explanation

Fig. 1 is the T-DNA district collection of illustrative plates of expression vector pCactF.LB and RB are respectively left margin and the right margin of T-DNA; Hyg represents hygromycin resistance; P35S represents the promotor of CaMV35S gene; T35S represents the terminator of CaMV35S gene; PAct1 represents the promotor of Actin gene; 3flag represents the flag sequence label of 3 times; OCS represents oCSthe terminator of gene; hindiII, kpnI, SpeI, xbai, sali and psti represents respectively the restriction enzyme site of restriction enzyme.

Fig. 2 is the Drought Stress Tolerance Analysis of A of KT525 transgenic line: A: arid is processed front seedling growth conditions; B: arid is processed rear seedling growth conditions; C: normally cultivate seedling growth conditions after a week; D: the survival rate statistics after arid is processed.

Fig. 3 is the soil Drought Stress Tolerance Analysis of A of KT525 transgenic line: A: drought is sieved front seedling growth conditions for the first time; B: drought is sieved rear seedling growth conditions for the first time; C: drought is sieved front seedling growth conditions for the second time; D: drought is sieved rear seedling growth conditions for the second time.

Fig. 4 is the Drought Stress Tolerance Analysis of A of KT525 transgenic line growth cycle: A: the growth conditions of seedling when rice plant drought sieve finishes; B: drought sieve finishes the state of rear Rice Panicle.

Fig. 5 is the salt size test of .KT525 transgenic line: A: salt sieve is processed front seedling growth conditions; B: salt sieve is processed rear seedling growth conditions; C: normally cultivate seedling growth conditions after a week; D: the survival rate statistics after salt sieve is processed.

Embodiment

In following embodiment, method therefor is ordinary method if no special instructions, the primer is synthetic by Shanghai Ying Jun biotech company, order-checking is completed by Beijing Hua Da gene, endonuclease in PCR test kit, vector construction process is purchased from precious biotechnology company limited, pEASY-T1 connects test kit purchased from Beijing Quan Shijin biotech company, T4 DNA ligase is purchased from Promega company, and the method that the equal reference reagent box of method provides is carried out.Carrier pHPG used in experiment is by this experimental reconstruction gained, and basic framework comes from the pCAMBIA1300 of CAMBIA company.

1, the separation of KT525 gene

From the coding initiation site ATG of KT525 gene, start to design 5 ' end primer, in terminator codon place design 3 ', hold primer:

Primer 1:5 ' GCACGCtctaga aTGAACCCCACCACCGCCGCC3 '

Primer 2: 5 ' GCACGCgtcgac tTATGGCTCGGCCTTCATGTGG3 '

In primer 1, tctaga sequence is the restriction enzyme site of restriction enzyme XbaI, and the sequence of underscore sign is kT525the encoding sequence of gene; In primer 2, gtcgac sequence is the restriction enzyme site of restriction enzyme SalI, and the sequence of underscore sign is the encoding sequence of KT525 gene.The sequence of primer 1 is as shown in SEQ ID No:3, and the sequence of primer 2 is as shown in SEQ ID No:4.

Extract total RNA of the fine paddy rice of Japan of Seedling Stage, by reverse transcription, obtain cDNA as template, total length with above primer amplification KT525 gene, its size is 792bp, its nucleotide sequence is as shown in SEQ ID NO:2 in sequence table, and coded protein amino acid sequence is as shown in the SEQ ID NO:1 in sequence table.

Concrete reaction is: the total RNA that gets approximately 2 μ g, add 1 μ l 10 * DNase buffer, the DNase of 1 μ l, mend DEPC treated water to 10 μ l system, mix, after 37 ℃ of incubation 30min, the RQ DNase stop solution that adds 1 μ l, 65 ℃ of incubation 10min with termination reaction after, add 2 μ l Oligo (dT) 18 primer (0.1 μ g/ μ l), 4 μ l 5 * First-strand buffer, 1 μ l Ribonuclease inhibitor (40U/ μ l), 2 μ l 4 * dNTP (each 10mM), 1 μ l MMLV Reverse Transcriptase (200U/ μ l), carefully mix, 37 ℃ are incubated 1 hour.Then process 5 minutes for 90 ℃, cooled on ice, centrifugal collection obtains corresponding reverse transcription product cDNA.By 10 times of the cDNA dilutions obtaining, get 1 μ l as the template of PCR reaction, each 1 μ l of upstream and downstream primer (10 μ M), LA Taq enzyme (5U/ μ l) 0.5 μ l, 4 * dNTPs (each 10mM), 1 μ l, 2 * GC buffer (Mg ²⁺) 25 μ l, H ₂o 20.5 μ l.PCR reaction conditions is 95 ℃ of 5min of preheating, 94 ℃ of 1min of sex change, and the 56 ℃ of 30sec that anneal, extend 72 ℃ of 1min50sec, 34 circulations.After reaction finishes, pcr amplification product is carried out to 1% agarose gel electrophoresis detection, obtained the DNA fragmentation that size conforms to expected results respectively.Reclaim respectively and the above-mentioned fragment of purifying, connect in carrier pEASY-T1 (Quan Shi King Company), by heat shock method, transform intestinal bacteria (E. coli) TOP10 bacterial strain, select positive bacterium colony and join 5ml containing in the LB liquid nutrient medium of 50mg/L kantlex, 37 ℃, in the shaking table of 200rpm, cultivate 12-16 hour, extract plasmid, obtain respectively the recombinant plasmid that contains object fragment, after sequence verification is correct, with XbaI and SalI double digestion, cut object fragment KT525, be connected into the pCactF plant expression vector that carries out identical double digestion, structure obtains carrier pCactF-KT525.Picking colony PCR is accredited as positive bacterium colony and carries out sequence verification.The T-DNA district collection of illustrative plates of plant expression vector pCactF as shown in Figure 1.

2. Agrobacterium cotransformation

With agrobacterium-mediated transformation, by the gene KT525 rice transformation relevant to resistance of reverse obtaining by embodiment 1, concrete grammar is as follows:

Utilize heat shock method that above-mentioned recombinant vectors pCactF-KT525 is proceeded to Agrobacterium AGL0 bacterial strain (Inst. of Genetics and Development Biology, CAS gives), utilize Agrobacterium to carry out cotransformation to paddy rice.

By the partial blade of the Transgenic Rice Plants obtaining, extract according to a conventional method total DNA, under the guiding of forward primer 5 '-ACTCACCGCGACGTCTGT-3 ' and reverse primer 5 '-TTCCTTTGCCCTCGGACG-3 ', pcr amplification hygromycin phosphotransferase gene, through amplification, obtain the positive transfer-gen plant of 828 bp DNA fragmentations, detected result shows to have obtained and transformed the transgenic paddy rice that has pCactF-KT525 with aforesaid method.

3, KT525 transgenosis T ₁drought Stress Tolerance Analysis of A for plant

3.1 KT525 transgenosis T ₁laboratory Drought Stress Tolerance Analysis of A for plant

The T1 of results KT525, for transgenic paddy rice seed, chooses 5 above strains and carries out arid screening.Its sprouting of 3 angels is cultivated in water seed soaking, then with 50mg/L Totomycin, carry out resistance screening, simultaneously to turn in the paddy rice of empty carrier, spend 11 to compare, screen after 5 days, adjoining tree is all dead, statistics transfer-gen plant resistance seedling and dead seedling number, the separated ratio of resistance of analysis transfer-gen plant, selects resistance seedling to be about the strain of 3: 1 with the separated ratio of non-resistant seedling, when seedling grows to 8cm left and right, seedling is transferred to from culture dish in triangular flask to every bottle of 10 strains, three repetitions.Be cultured to tri-leaf period (the 3rd leaf unfolded completely), its growth conditions is as Fig. 2 A.Use the Hoagland solution that contains 20% PEG instead and carry out drought sieve, approximately 3 days, there is blade tip jaundice whiting in contrast, when blade is sagging or partly volume is rolled up even entirely (Fig. 2 B), washes away PEG solution, normally cultivates.Within every 1.5 days during this time, change a PEG solution.Nutritive medium in triangular flask is changed once for two days, to keep the fresh state of nutritive medium.Take a picture (Fig. 2 C) after one week, statistics survives seedling number, calculates drought-enduring seedling ratio, result as Fig. 2 D(X-coordinate be different transgenic line numberings, ordinate zou is drought-enduring seedling per-cent).Growth conditions and data statistics by seedling can learn that KT525 transfer-gen plant has drought tolerance.

3.2 laboratory soil drought size test

Except above-mentioned screening, also carried out soil drought sieve, the seedling that is cultured to tri-leaf period is transferred in small flower, Adaptable growth one week.Seedling waters to saturated, removes excessive moisture, and arid is processed approximately two weeks, and the contrast rehydration that occurs taking a picture after leaf rolling is processed, with twice of same method drought sieve.Experimental result is shown in Fig. 3, after arid is processed, and KT525 transgenic paddy rice T ₁can continued growth after the generation plant renewal cultivation of positive strain, flower 11(ZH11 in wild-type contrast) and turn the yellow leaf, curling, withered of empty carrier contrast (469) plant, cane can not be upright, death (Fig. 3 D) very soon after renewal cultivation.Twice drought sieve result of soil shows, adjoining tree leaf rolling, jaundice, withered under equal conditions, and KT525 transfer-gen plant well-grown (Fig. 3 B, D).

The Drought Stress Tolerance Analysis of A of 3.3 KT525 transgenic line growth cycles

We by meet resistance than the seedling replanting of 3:1 to as cultivated in the container of Fig. 4 A, plant to be planted grows into that the heading-stage starts arid until growth finishes.From experimental result, the KT525 transfer-gen plant that do not water under field conditions (factors) has more drought tolerance (Fig. 4 A, 4B) than control group paddy rice.The above results shows T ₁for transgenic rice plant to arid tolerance apparently higher than transgenic rice plant not.

4, KT525 transgenosis T1 is for the Salt Tolerance Analysis of plant

Results KT525 T1, for transgenic paddy rice seed, chooses 5 above strains and carries out salt stress.Its sprouting of 3 angels is cultivated in water seed soaking, then with 50mg/L Totomycin, carry out resistance screening, simultaneously to turn in the paddy rice of empty carrier, spend 11 to compare, screen after 5 days, adjoining tree is all dead, statistics transfer-gen plant resistance seedling and dead seedling number, the separated ratio of resistance of analysis transfer-gen plant, selects resistance seedling to be about the strain of 3: 1 with the separated ratio of non-resistant seedling, when seedling grows to 8cm left and right, seedling is transferred to from culture dish in triangular flask to every bottle of 10 strains, three repetitions.Be cultured to tri-leaf period (the 3rd leaf unfolded completely), its growth conditions is as Fig. 5 A.Use the Hoagland solution that contains 200mM NaCl instead and carry out salt sieve, about approximately 3 days, there is blade tip jaundice whiting in contrast, when blade is sagging or partly volume is rolled up even entirely (Fig. 5 B), washes away salts solution, normally cultivates.Within every 1.5 days during this time, change a salts solution.Rehydration second day is changed one time of nutrition liquid again, to remove remaining salinity.Nutritive medium in triangular flask is changed once for 2 days, to keep the fresh state of nutritive medium.Take a picture (Fig. 5 C) after one week, statistics survives seedling number, calculates salt tolerant seedling ratio, result as Fig. 5 D(X-coordinate be different transgenic line numberings, ordinate zou is salt tolerant seedling per-cent).Experimental result shows, T1 for transgenic rice plant to the tolerance of salt apparently higher than transgenic rice plant not, after salt is processed, KT525 transgenic paddy rice T1 can continued growth after for the plant renewal cultivation of positive strain, turn empty carrier in spend the yellow leaf, curling, withered of 11 contrasts (CK) plant, cane can not be upright, after renewal cultivation very fast dead (Fig. 5 C).

This description of test KT525 gene has the effect that strengthens Salt Resistance of Rice.

SEQUENCE LISTING

<110> Beijing Weiming Kaituo Crops Design Center Ltd

Gene KT525 of <120> is in the application improving on plant stress tolerance

<130>

<150> CN201210495569.0

<151> 2012-11-28

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 263

<212> PRT

<213> Oryza sativa L.ssp.japonica

<400> 1

Met Asn Pro Thr Thr Ala Ala Ala Ala Asp Gln Pro Ser Lys Pro Ser

1 5 10 15

Ala Ala Ala Ala Ala Arg Lys Arg Lys Ser Ser Ala Lys Pro Lys Ala

20 25 30

Ser Ser Ser Ser Leu Pro Thr Ala Thr Ala Thr Thr Asn Ala Ser Pro

35 40 45

Lys Arg Ser Lys Val Ala Ala Gly Ala Gly Asp Asp Gly Asp Ala Asp

50 55 60

Ala Asp Ala Ala Glu Glu Lys Pro Glu Pro Ala Lys Asp Tyr Ile His

65 70 75 80

Val Arg Ala Arg Arg Gly Gln Ala Thr Asp Ser His Ser Leu Ala Glu

85 90 95

Arg Val Arg Arg Glu Arg Ile Ser Glu Arg Met Lys Leu Leu Gln Ser

100 105 110

Leu Val Pro Gly Cys Asn Lys Ile Thr Gly Lys Ala Leu Met Leu Asp

115 120 125

Glu Ile Ile Asn Tyr Val Gln Ser Leu Gln Arg Gln Val Glu Phe Leu

130 135 140

Ser Met Lys Leu Ala Thr Met Asn Pro Gln Leu Asp Phe Asp Ser His

145 150 155 160

Tyr Met Pro Ser Lys Asp Met Ser His Met Pro Val Pro Ala Tyr Pro

165 170 175

Ser Ser Asp Pro Thr Thr Thr Thr Ala Phe Ser Tyr Thr Gly Ser Pro

180 185 190

Ala Thr Ala Asp Pro Phe Thr Val Tyr Asn Cys Trp Glu Leu Asp Leu

195 200 205

His Thr Ala Met Gln Met Gly Ala Thr Thr Gly Leu Ser Gln Asp Gly

210 215 220

Pro Ile Ala Thr Met Ala Pro Ser Pro Ser Pro Leu Pro His His Pro

225 230 235 240

Pro Leu His Gly Phe Tyr Gly Gly Gln Gln Gln Gln Gly Thr Thr Val

245 250 255

Asn His Met Lys Ala Glu Pro

260

<210> 2

<211> 792

<212> DNA

<213> Oryza sativa L.ssp.japonica

<400> 2

atgaacccca ccaccgccgc cgccgccgac caaccatcca agccctccgc cgccgccgcc 60

gcccggaagc gcaagtcgtc ggcgaagccc aaggcctcgt cctcatcctt acccacggcc 120

acggcgacga cgaacgcgag cccgaagcgg tccaaggtcg ccgccggcgc cggagacgac 180

ggcgacgccg acgccgacgc ggcggaggag aagccggagc cagccaaaga ctacatccat 240

gtgagggcga ggcgggggca agccaccgat agccatagcc tcgccgagag ggtgaggagg 300

gagaggataa gcgagaggat gaagcttctg cagtcgctcg tgccaggctg caacaagatc 360

accggcaagg ctctcatgct ggacgagatc atcaactatg tgcagtcgct gcagcgtcag 420

gtcgagtttt tgtccatgaa gttggcgacc atgaatcctc agctggactt tgacagccat 480

tacatgcctt ccaaagatat gagccatatg ccagtacccg catacccgtc aagcgatccg 540

accaccacca ccgcgttctc ctacaccggc tcacccgcca ctgctgatcc attcaccgtc 600

tacaactgct gggagctcga cctccacacc gctatgcaaa tgggagccac caccggactc 660

agccaagacg gtccaatcgc aacgatggca ccctctccct cgccattgcc gcaccatcct 720

cctcttcacg gcttctacgg ggggcagcag cagcagggga cgacagtaaa ccacatgaag 780

gccgagccat aa 792

<210> 3

<211> 33

<212> DNA

<213> artificial sequence

<400> 3

gcacgctcta gaatgaaccc caccaccgcc gcc 33

<210> 4

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gcacgcgtcg acttatggct cggccttcat gtgg 34

Claims (15)

1. a gene, is characterized in that, the nucleotide sequence of described gene is as shown in SEQ ID NO:2 in sequence table.

2. the expression vector, the expression cassette that contain gene described in claim 1.

3. the protein of genes encoding claimed in claim 1.

4. protein according to claim 3, is characterized in that, the aminoacid sequence of described protein is as shown in SEQ ID NO:1.

5. the application of the protein of gene claimed in claim 1 and coding thereof in cultivating resistance of reverse transgenic plant.

6. application claimed in claim 5, described resistance of reverse is salt tolerance and/or drought tolerance.

7. the application described in claim 5 or 6, it is characterized in that: the resistance to contrary gene of plant is inserted to expression vector, the plant expression vector that acquisition contains the resistance to contrary gene of plant, then this plant expression vector is imported to object plant, from expressing the plant screening of described plant stress tolerance, obtain the plant that resistance of reverse strengthens.

8. the application described in claim 5-7 any one, is characterized in that: described plant is dicotyledons or monocotyledons, is preferably monocotyledons, is especially preferably paddy rice.

9. application claimed in claim 8, the feature of wherein said expression vector is: for building the carrier that sets out of described plant expression vector, be a kind of carrier that can be used for the binary vector of agrobacterium tumefaciens or Agrobacterium rhizogenes conversion of plant or can be used for plant micropellet bombardment, or the carrier that can copy in prokaryotic organism.

10. application claimed in claim 8, it is characterized in that: bringing Selection In property marker gene in described plant expression vector, described selected marker includes but not limited to: the coding that can express in plant can produce the enzyme of colour-change, the gene of luminophor, the antibiotic marker thing with resistance, anti-chemical reagent marker gene.

11. application claimed in claim 9, the wherein said carrier that sets out is pCAMBIA serial carrier.

Application described in 12. claims 11, the wherein said carrier that sets out is pCAMBIA1300.

13. 1 kinds of methods of cultivating transgenic plant, are that gene claimed in claim 1 is imported in object plant, obtain resistance of reverse higher than the transgenic plant of described object plant.

14. methods according to claim 13, is characterized in that: described resistance of reverse is salt tolerance and/or drought tolerance.

15. according to the method described in claim 13 or 14, it is characterized in that: described object plant is dicotyledons or monocotyledons, is preferably monocotyledons, is especially preferably paddy rice.

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