CN114015666B

CN114015666B - Application of OsPARP3 gene in regulation and control of plant drought tolerance

Info

Publication number: CN114015666B
Application number: CN202111322839.3A
Authority: CN
Inventors: 李秀梅; 刘勤坚
Original assignee: Agro-Biological Gene Research Center Guangdong Academy Of Agricultural Sciences
Current assignee: Agro-Biological Gene Research Center Guangdong Academy Of Agricultural Sciences
Priority date: 2021-11-09
Filing date: 2021-11-09
Publication date: 2022-08-12
Anticipated expiration: 2041-11-09
Also published as: CN114015666A

Abstract

The invention discloses application of an OsPARP3 gene in regulation and control of plant drought tolerance. The invention proves that the rice OsPARP3 gene is a functional gene for rice drought resistance for the first time, and the gene responds to drought stress and ABA induction of abscisic acid; the drought stress tolerance of the gene mutant plant is lower than that of a wild type, and the drought stress tolerance of the over-expression plant is obviously higher than that of the wild type. The OsPARP3 gene can be used for improving the drought tolerance of the existing gramineous plants such as rice and the like through the technologies of molecular marker assisted breeding, genetic transformation and the like, and has wide application prospect in cultivating and creating intermediate materials and production varieties with high drought tolerance.

Description

Application of OsPARP3 gene in regulation and control of plant drought tolerance

Technical Field

The invention belongs to the technical field of plant resistance breeding, and particularly relates to application of an OsPARP3 gene in regulation and control of plant drought tolerance.

Background

Drought stress can significantly affect the growth and development of plants and also plays a crucial role in plant distribution pattern. Drought stress responses include osmoregulatory genes, antioxidant metabolism genes, stress-inducing protein genes. The phytohormone abscisic acid (ABA) plays a role in the adaptive response of plants to abiotic stressThe important function is a key regulatory factor for regulating gene expression in a stress adaptation signal transduction pathway. When the environment is normal, the abscisic acid content in the plant body is extremely low. When suffering from drought, the abscisic acid content is increased, the ion flux of protective cells is regulated, stomata are mediated to be closed, and the water loss is reduced. Mediation of activation of protein kinase SnRK2.6/OST1 and other signaling mechanisms such as ROS and Ca ²⁺ . Exogenous ABA is added during drought stress, so that the content of proline and soluble protein in a plant body can be obviously increased, and the damage of the drought stress to leaves is effectively relieved, so that the physiological state of the leaves is improved, and the normal growth of the plant is maintained. As is well known, rice is a drought susceptible crop and requires a large amount of water during the growth process. Its root system is small, stomata are closed quickly, and the waxy output of stratum is reduced under the condition of slight water stress. Therefore, the new rice variety with drought resistance is cultivated, and the healthy growth of the rice can be promoted while the labor amount is reduced. Introduction of stress-activated responsive genes into rice to enhance drought resistance is an effective way to increase rice yield and quality under increasingly severe environmental stress; the agricultural yield is higher, and meanwhile, the method is environment-friendly and realizes the aim of sustainable development of agriculture.

Poly ADP-ribose polymerase is involved in a series of biological events such as DNA damage response, stress repair, transcription and posttranscriptional regulation and cell programmed death. PARP is an important signaling pathway for injury repair pathways, recruiting other necessary repair proteins to maintain genomic integrity. In addition, there are a series of reports that PARP is a positive regulator of plant immune response. The rice genome contains 3 PARP family members, and the OsPARP3 gene is highly expressed in the mature dehydration stage of rice seeds. However, the research and application of the gene in the aspect of regulating and controlling the drought tolerance of rice are not reported.

Disclosure of Invention

The invention aims to provide application of a rice OsPARP3 gene in drought resistance regulation. The OsPARP3 gene is cloned from a rice model material Nipponbare, and the OsPARP3 gene is transferred into rice to improve the expression level of the OsPARP3 gene, so that the drought stress tolerance of the rice is obviously improved. Therefore, the overexpression of the OsPARP3 gene in rice has important significance for improving the drought resistance of rice.

The invention is based on a rice T-DNA insertion mutant with reduced drought resistance, PCR amplification is carried out by specific primers to verify the insertion position of the insertion mutant, and the inserted specific gene is a gene number Os02g0530600 and an OsPARP3 gene for coding PARP protein. The rice OsPARP3 gene sequence has a full length of 2496bp and encodes 831 amino acids. Then, taking a Nipponbare plant as a material, extracting total RNA of the Nipponbare plant, and then reversely transcribing the RNA into cDNA. And (3) designing a primer by taking the reverse transcription product as a template to carry out PCR amplification, recovering a target fragment and cloning the whole gene fragment. An overexpression vector fused with the OsPARP3 gene is introduced into Nipponbare through an agrobacterium EHA105 mediated genetic transformation method. The transgenic plant is identified by PCR and quantitative PCR, which proves that the target gene is transformed into rice and the expression level of the target gene OsPARP3 is improved. And (4) carrying out selfing propagation on the over-expression transgenic positive plants to obtain homozygous positive transgenic lines. OsPARP3 is induced by drought stress and ABA, and corresponding to the expression mode, the drought resistance of over-expressed rice plants is obviously higher than that of wild plants. These results indicate that OsPARP3 is a positive regulator of rice ABA signaling and drought stress tolerance. The OsPARP3 has high application potential in rice drought resistance genetic improvement.

Therefore, the first objective of the invention is to provide a poly ADP-ribose polymerase, the amino acid sequence of which is shown as SEQ ID NO. 2.

It is a second object of the present invention to provide a poly ADP-ribose polymerase gene encoding the poly ADP-ribose polymerase.

Preferably, the poly ADP-ribose polymerase gene is an OsPARP3 gene, and the nucleotide sequence of the poly ADP-ribose polymerase gene is shown as SEQ ID NO. 1.

The third purpose of the invention is to provide a recombinant vector containing the poly ADP-ribose polymerase gene (OsPARP3 gene).

Preferably, the recombinant vector is a vector for over-expressing a poly ADP-ribose polymerase gene (OsPARP3 gene).

The fourth object of the present invention is to provide a recombinant strain or a recombinant cell containing the poly ADP-ribose polymerase gene (OsPARP3 gene).

The fifth purpose of the invention is to provide the application of the poly ADP-ribose polymerase gene (OsPARP3 gene) in regulating and controlling the drought tolerance of plants.

Preferably, the application is the application of overexpression poly ADP-ribose polymerase gene (OsPARP3 gene) in enhancing plant drought tolerance.

Preferably, the plant is a gramineous plant.

Preferably, the plant is rice.

The invention also comprises DNA fragments which are homologous with the OsPARP3 gene, so long as the coded proteins are the same or similar to the displayed proteins in the aspects of biological functions, physiological and biochemical characteristics and the like, and can respond to ABA induction and increase drought resistance by up-regulating the expression quantity and/or activity displayed by OsPARP 3. These DNA fragments homologous to the OsPARP3 gene include alleles, homologous genes, mutant genes and derivative genes corresponding to the nucleotide sequence of the present invention; the proteins encoded by the protein are similar to the protein of the invention, or the phenomenon of substitution, deletion or insertion of one, several or tens of amino acids exists, and the protein belongs to the content of the invention.

The invention has the advantages that:

1. provides a gene OsPARP3 for improving the drought stress tolerance of rice, and over-expresses the OsPARP3 gene in the rice for the first time. The drought stress tolerance of the over-expressed OsPARP3 gene positive plant is obviously improved. Based on the newly discovered OsPARP3 gene, the gene can be used for improving the drought tolerance of gramineous plants such as rice and the like, and cultivating and creating intermediate materials and production varieties with high drought tolerance by the technologies of molecular marker assisted breeding, genetic transformation and the like.

2. The OsPARP3 gene is related to rice resistance, responds to hormone ABA induction, provides a material for researching and controlling the interaction regulation of hormone and adversity, is also beneficial to disclosing a molecular mechanism of the OsPARP3 gene of rice for improving the drought stress tolerance of rice, and has wide application prospect for safe production of rice and labor force reduction.

Drawings

FIG. 1 shows the identification and isolation of T-DNA insertion mutants; wherein: FIG. A: schematic representation of the insertion position of the mutant rice T-DNA, the square boxes are marked as coding regions, the straight lines are expressed as introns, and the triangles show the insertion position of the T-DNA; and B: sequencing a peak image; and (C) figure: schematic diagram of PCR detection of Osparp3 mutant rice line separation and identification; from left to right, the first lane is a wild type Oryza sativa L.ssp.Japonica cv.Donjin plant control, the second lane is a heterozygous mutant, the third lane is a homozygous mutant, the upper DNA band is subjected to amplification by an OsPARP3 genome specific primer, and the lower DNA band is subjected to combined amplification by a genome and an inserted T-DNA specific primer; FIG. D: the western-blot assay showed a loss of PARP3 protein expression in the mutants.

FIG. 2 is a map of overexpression vector pXU 1301.

Fig. 3 shows the expression level of osprp 3 in transgenic plants over-expressed in this genetic background and Oryza sativa l.

FIG. 4 is the expression level of OsPARP3 after drought and ABA treatment; panel A is drought treatment (PEG 6000); panel B is ABA treatment.

FIG. 5 shows that overexpression of OsPARP3 increases drought resistance in rice, and in FIG. A shows the phenotype of rice after drought treatment, wherein the scale: 5 cm; DJ denotes Oryza sativa l.ssp.jannica cv.donjin, mutan denotes a homozygous mutant OsPARP3 strain in the background of DJ, Nip denotes Oryza sativa l.ssp.janica cv.nipponbare, OE6, OE10, OE19 are osprp 3 homozygous over-expressed rice strains in the background of Nip; panel B shows the survival of the corresponding drought-treated rice, with one star indicating a significant difference in the mutant versus the control DJ and two stars indicating a very significant difference versus the control Nip.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

The technical solution of the present application will be described in detail with reference to specific examples. The test reagents and consumables used in the examples of the present invention are conventional test materials in the art and are commercially available.

Example 1: identification of T-DNA insertion mutants

The invention is based on a rice (Oryza sativa L.ssp. Japonica cv.Donjin) T-DNA insertion mutant with reduced drought tolerance, and PCR amplification is carried out by specific primers to verify that the insertion position of the insertion mutant and the inserted specific gene are a gene number Os02g0530600 and a gene OsPARP3 coding PARP protein. The invention obtains homozygous T-DNA insertion mutant rice by identification and separation, and further verifies the expression deletion of OsPARP3 in the mutant by using western-blot.

Planting the obtained T-DNA mutant rice, sampling leaves in a single plant, extracting the genome DNA of the mutant leaves, amplifying by using a boundary sequence primer of a T-DNA carrier and a primer matched with a flanking sequence of the OsPARP3 gene, and sequencing. Genomic sequences can be amplified using primers LP and RP, and insertion of T-DNA into genomic sequences can be detected using primers RB1 and LP. Only lines with a single amplified band matched with the RB1+ LP primers were homozygous lines (FIG. 1). The primer sequences involved are as follows: RB 1: CCACAGTTTTCGCGATCCAGACTG, respectively; and (3) LP: TATCGCCTCCTAAATCGCAC, respectively; RP: GGTCAGCTCGAGTGTAAGGG are provided. Progeny can be further propagated by identifying the obtained homozygous mutant osparp 3. Polyclonal rabbit antibodies are prepared by using the OsPARP3 gene sequence fragment, and the expression deletion of the OsPARP3 in the mutant is verified again through the extraction of total protein, the SDS-PAGE electrophoresis of protein, membrane transfer and antibody incubation.

Example 2: cloning of OsPARP3 gene and construction of overexpression plant

1. Taking a model rice material Nipponbare (Oryza sativa L.ssp.Japonica cv.Nipponbare) seed, and carrying out RNA extraction according to the instruction of a Beatle polysaccharide polyphenol RNA plant extraction kit (containing DNA enzyme); the purity and concentration of total RNA are detected by agarose gel electrophoresis and a nanodrop microspectrophotometer, 1 mu g of total RNA is taken for carrying out the initial reverse transcription reaction, the adopted reverse transcriptase is PrimeScript, and the steps of the reverse transcription reaction refer to the use instruction of the reverse transcriptase. Taking a reverse transcription product as a template, and adopting a primer F: ATGGTACACGAGACAAGATCACG, respectively; r: TCACTCGTCCGGCACCAC, PCR amplification was performed using KOD FX (Toyobo) as the polymerase. The reaction system was 50uL, and the PCR reaction system was prepared according to the instructions of KOD FX. The reaction conditions are as follows: 5min at 94 ℃; 30 cycles of 94 ℃ 30sec, 58 ℃ 30sec, 68 ℃ 150 sec; 10min at 68 ℃. PCR amplification yielded a fragment of about 2500 bp. The fragment is recovered by a method of directly purifying a PCR product, and then the fragment is connected with a T vector and transferred into Escherichia coli DH5 alpha. And (4) picking clones for sequencing. The total length of CDS sequence of the OsPARP3 gene is 2496bp, and the nucleotide sequence is shown as SEQ ID NO. 1; the coded amino acid sequence of the gene is shown as SEQ ID NO. 2.

2. The CDS of the OsPARP3 gene amplified by the above PCR was ligated to an overexpression vector pXU1301 (vector map is shown in FIG. 2) after cutting out GUS exon2 with HindIII and BamH I double enzymes by homologous recombination. After transformation, 5 monoclonals are selected for plasmid extraction, enzyme digestion identification is carried out, and two positive clones are selected for sequencing to determine that the plasmid construction is correct. And (3) transforming the correct plasmid into wild type Nipponbare callus by using agrobacterium EHA105, screening callus with resistance through pre-culture, infection and co-culture, differentiating, rooting, hardening seedlings and transplanting to obtain a transgenic plant. And transferring the screened positive plants to a planting pot, and collecting seeds after the positive plants are mature to obtain T1 generation plants. 5 plants which are detected to be positive by PCR are selected from each strain for selfing and breeding to obtain T2 generation plants, and the T2 generation strains are further detected by PCR to obtain OsPARP3 homozygous over-expression strains.

3. The ears of 3 strains in the booting stage are taken to carry out fluorescence quantitative PCR, and the wild Nipponbare is taken as a control to detect the expression level of the target gene OsPARP 3. Extracting total RNA by using TriZol Reagent, carrying out reverse transcription on the cDNA first strand to synthesize and calculate the required volume of 1 mu g of RNA template, mixing the RNA template with a solution of a kit, slightly shaking and uniformly mixing the RNA template and the solution, and carrying out short centrifugation at 37 ℃ for 5min to remove residual DNA of an RNA genome; after the above reaction, first strand cDNA synthesis was performed, and the above reaction solution was centrifuged by adding the reagent in the kit to the above reaction solution for a short time, and then the mixture was kept at 42 ℃ for 15min and heated at 85 ℃ for 5min until inactivation of Star ScriptIIRT Mix, to obtain a cDNA solution. After the reaction is completed, diluting the obtained cDNA by 10 times; the upstream and downstream primers are respectively as follows: f: CGTTCAACTTTAGATGACCCACTG, R: CCTTGACAGGCTCGTAGGTTTTC, reference gene actin (F: CGGTGTCATGGTCGGAAT, R: GCTCGTTGTAGAAGGTGT) of rice was used as a control. The results are shown in FIG. 3, and the expression level of OsPARP3 of 3 over-expressed transgenic lines is obviously improved by 35-60 times.

Example 3: functional identification of OsPARP3 gene

1. Expression of OsPARP3 at mRNA level after ABA and drought treatment of rice

The roots of the leaves of the Japanese fine 3 are respectively immersed into a rice culture solution containing 5 MuM ABA and 20% PEG6000 for treatment, and materials with different treatment time lengths of 0, 1, 3, 6, 12 and 24h are taken. Total stem and leaf RNA was extracted using TriZol Reagent, and the assay was performed as described in step 3 of example 2. The results are shown in figure 4, the OsPARP3 gene in both stems and roots was induced to be up-regulated under ABA and drought treatment (PEG6000 treatment), indicating that the OsPARP3 gene responds to ABA and drought.

2. OsPARP3 overexpression transgenic lines and mutant drought treatment

Harvested OsPARP3 homozygous over-expression lines, homozygous mutant ospparp 3 lines and wild type DJ, wild type Nipponbare seeds were germinated separately in light incubators at 28 ℃. After 2 days, the germinated seedlings were transferred to a rice culture solution, and after about 2 weeks, seedlings of respective lines having substantially uniform growth conditions were treated with 20% PEG6000 in a light incubator at 28 ℃ for 7 days, at least 45 plants per line, and then rehydrated for 3 days. The experiment was repeated three times to count the survival rate of each strain. The results are shown in fig. 5, where the homozygous mutant ospparp 3 plants had reduced drought stress tolerance and 0 plant survival compared to wild type DJ plants. The survival rate of OsPARP3 homozygous over-expression plants (OE6, OE10 and OE19) is obviously higher than that of wild Nipponbare, and the plants are drought-resistant.

Sequence listing

<110> center for researching agricultural biological genes of Guangdong province academy of agricultural sciences

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2496

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 1

atggtacacg agacaagatc acgcacacta gcggcttccc aagaagaagg gaaggctgct 60

ccgaagaagc agaagacaga aagcaaagag caggaaggtg gccagcaagc gccctccaag 120

aacaagaaga cagccgacaa tgaagagcac gacggggagc aggagccatc aaagaacaag 180

aagctcaaag cggaggaatc tgacctgaat gggaaggcaa cagccgtgaa ggagttctct 240

gaattctgca aggccataag ggaacacctc acgatcgagg acatgcgcaa gatcctccaa 300

ggcaatgaac aggatgcatc tggatcagaa gatgcagttg tcccaagatg tgaggatgtg 360

atgttttatg ggcctcttga caagtgcccc gtatgcggtg gtcagctcga gtgtaaggga 420

ttgaaataca attgcactgg gactcacagt gagtgggcat gttgtagctt cagtactaac 480

aatccttcga ggagaggtgg tcctataaag gtccctgatg atgtcaagaa tgatttcgtg 540

cgcaagtggc tgaagcaaca agaagggaat aagtacccta aacgtaattt ggatgacgag 600

ggtatcttct cgggcatgat gattgcattg tctggaagga tgtcacgctc acatggttat 660

ttcaaggagc agattatgaa gcatggaggg aaagtcaaca actccgtgat tggtgtcact 720

tgtgtagtgg cttccccagc tgagagacat caaggtggct ctggaggatt tgccgaagca 780

ctggagcgtg gaactcctgt tgtgagtgag aattggataa ttgatagcgt ccagaagaag 840

gaaaagcaac ctttggctgc ttatgatatt gcatctgatg ttgttccaga aggccgaggg 900

ctgccgctgg gcaaccttga tccaactgag gaggctatcg agaccttggc tgcagagctt 960

aaacttgcag gcaaaagagc agtgcacaaa gattctaaac tggagaaaga cggaggacat 1020

atctatgaga aggatggcat catctacaat tgtgcctttt ctgtgtgcga tttaggaggc 1080

gatattaacc agttatgcat tatgcagctg atcatggtgc ctgagaacca tttgcaccta 1140

tactacaaga agggtcctat tggacatgac cagatggcag aggaacgggt tgaagatttt 1200

ggtagtcgtt tcaatgatgc tatcaaggaa tttgtccgtc tttttgagga ggttactgga 1260

aatgagtttg aaccatggga gagagagaag aaatttaaga agaagtgtat gaagatgtat 1320

cccttggaca tggatgatgg tgttgacgtg cgccatggtg gtgtggctct tcgtcagttg 1380

ggagctgctg cagcacactg caagcttgac ccttctgtta cttttatcat gaaacaacta 1440

tgcagccaag aaatatacag gtatgctttg acagagatgg gccatgatgt gccagacctt 1500

cctatcggga tgcttacaga tctccatctg aaaagagggg aggagacgct actagagtgg 1560

aaacaagatg tcgagtctgc tccagagtct gggcctgctg ctgacgcatt ctggatggag 1620

atcagcaata agtggttcac cctattcccc acaactcgtc catatacaat gaagggatat 1680

gaacagattg ctgacaatgt ggcttctggt ttggagactg ttcgtgatat aaatgtcgct 1740

tctcgtctta ttggtgatgt gtttggttca actttagatg acccactgtc tcaatgctac 1800

aagaaactag gctgttctat caatcgtgtt gttgaagact cggaggacta taagatgatc 1860

ctgaagtatc tggagaaaac ctacgagcct gtcaaggttg gcgatgtggt ttacagtgct 1920

actgtcgagc ggatatatgc tgttgaatcg agtgctcttc cttcttatga tgaaataaag 1980

aaacttccga acaaagttct cctctggtgt ggaacaagaa gttcaaattt gctcaggcac 2040

ctgcgcgatg gtttcgtgcc tgctgtctgc catataccag tgccgggata catgtttggc 2100

aaagccatag tttgctcaga cgcagcagcc gaagccgcgc tctacggctt cacggcggtg 2160

gatcgtccgg agggttacct ggtcctggcg gtggcatctc tggggaaaga gatccaggag 2220

ataacaggca cgccaggctc agaggacgtg aaacgcatgg aggagaagaa gatgggcgtg 2280

aagggagtcg ggaggaagac gaccgacccg tcggagcact tcacctggag ggacggcgtc 2340

acggtgccgt gcggcaagct ggtgccgtcc acgaacaagg acggcccgct cgagtacaac 2400

gagtacgccg tgtacgaccc caagcaggtg agcatcgcgt tcctcgtggg cgtgaagtac 2460

gaggagcaga acatggaggt ggtgccggac gagtga 2496

<210> 2

<211> 831

<212> PRT

<213> Rice (Oryza sativa L.)

<400> 2

Met Val His Glu Thr Arg Ser Arg Thr Leu Ala Ala Ser Gln Glu Glu

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Gly Lys Ala Ala Pro Lys Lys Gln Lys Thr Glu Ser Lys Glu Gln Glu

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Gly Gly Gln Gln Ala Pro Ser Lys Asn Lys Lys Thr Ala Asp Asn Glu

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Glu His Asp Gly Glu Gln Glu Pro Ser Lys Asn Lys Lys Leu Lys Ala

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Glu Glu Ser Asp Leu Asn Gly Lys Ala Thr Ala Val Lys Glu Phe Ser

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Glu Phe Cys Lys Ala Ile Arg Glu His Leu Thr Ile Glu Asp Met Arg

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Lys Ile Leu Gln Gly Asn Glu Gln Asp Ala Ser Gly Ser Glu Asp Ala

100 105 110

Val Val Pro Arg Cys Glu Asp Val Met Phe Tyr Gly Pro Leu Asp Lys

115 120 125

Cys Pro Val Cys Gly Gly Gln Leu Glu Cys Lys Gly Leu Lys Tyr Asn

130 135 140

Cys Thr Gly Thr His Ser Glu Trp Ala Cys Cys Ser Phe Ser Thr Asn

145 150 155 160

Asn Pro Ser Arg Arg Gly Gly Pro Ile Lys Val Pro Asp Asp Val Lys

165 170 175

Asn Asp Phe Val Arg Lys Trp Leu Lys Gln Gln Glu Gly Asn Lys Tyr

180 185 190

Pro Lys Arg Asn Leu Asp Asp Glu Gly Ile Phe Ser Gly Met Met Ile

195 200 205

Ala Leu Ser Gly Arg Met Ser Arg Ser His Gly Tyr Phe Lys Glu Gln

210 215 220

Ile Met Lys His Gly Gly Lys Val Asn Asn Ser Val Ile Gly Val Thr

225 230 235 240

Cys Val Val Ala Ser Pro Ala Glu Arg His Gln Gly Gly Ser Gly Gly

245 250 255

Phe Ala Glu Ala Leu Glu Arg Gly Thr Pro Val Val Ser Glu Asn Trp

260 265 270

Ile Ile Asp Ser Val Gln Lys Lys Glu Lys Gln Pro Leu Ala Ala Tyr

275 280 285

Asp Ile Ala Ser Asp Val Val Pro Glu Gly Arg Gly Leu Pro Leu Gly

290 295 300

Asn Leu Asp Pro Thr Glu Glu Ala Ile Glu Thr Leu Ala Ala Glu Leu

305 310 315 320

Lys Leu Ala Gly Lys Arg Ala Val His Lys Asp Ser Lys Leu Glu Lys

325 330 335

Asp Gly Gly His Ile Tyr Glu Lys Asp Gly Ile Ile Tyr Asn Cys Ala

340 345 350

Phe Ser Val Cys Asp Leu Gly Gly Asp Ile Asn Gln Leu Cys Ile Met

355 360 365

Gln Leu Ile Met Val Pro Glu Asn His Leu His Leu Tyr Tyr Lys Lys

370 375 380

Gly Pro Ile Gly His Asp Gln Met Ala Glu Glu Arg Val Glu Asp Phe

385 390 395 400

Gly Ser Arg Phe Asn Asp Ala Ile Lys Glu Phe Val Arg Leu Phe Glu

405 410 415

Glu Val Thr Gly Asn Glu Phe Glu Pro Trp Glu Arg Glu Lys Lys Phe

420 425 430

Lys Lys Lys Cys Met Lys Met Tyr Pro Leu Asp Met Asp Asp Gly Val

435 440 445

Asp Val Arg His Gly Gly Val Ala Leu Arg Gln Leu Gly Ala Ala Ala

450 455 460

Ala His Cys Lys Leu Asp Pro Ser Val Thr Phe Ile Met Lys Gln Leu

465 470 475 480

Cys Ser Gln Glu Ile Tyr Arg Tyr Ala Leu Thr Glu Met Gly His Asp

485 490 495

Val Pro Asp Leu Pro Ile Gly Met Leu Thr Asp Leu His Leu Lys Arg

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Gly Glu Glu Thr Leu Leu Glu Trp Lys Gln Asp Val Glu Ser Ala Pro

515 520 525

Glu Ser Gly Pro Ala Ala Asp Ala Phe Trp Met Glu Ile Ser Asn Lys

530 535 540

Trp Phe Thr Leu Phe Pro Thr Thr Arg Pro Tyr Thr Met Lys Gly Tyr

545 550 555 560

Glu Gln Ile Ala Asp Asn Val Ala Ser Gly Leu Glu Thr Val Arg Asp

565 570 575

Ile Asn Val Ala Ser Arg Leu Ile Gly Asp Val Phe Gly Ser Thr Leu

580 585 590

Asp Asp Pro Leu Ser Gln Cys Tyr Lys Lys Leu Gly Cys Ser Ile Asn

595 600 605

Arg Val Val Glu Asp Ser Glu Asp Tyr Lys Met Ile Leu Lys Tyr Leu

610 615 620

Glu Lys Thr Tyr Glu Pro Val Lys Val Gly Asp Val Val Tyr Ser Ala

625 630 635 640

Thr Val Glu Arg Ile Tyr Ala Val Glu Ser Ser Ala Leu Pro Ser Tyr

645 650 655

Asp Glu Ile Lys Lys Leu Pro Asn Lys Val Leu Leu Trp Cys Gly Thr

660 665 670

Arg Ser Ser Asn Leu Leu Arg His Leu Arg Asp Gly Phe Val Pro Ala

675 680 685

Val Cys His Ile Pro Val Pro Gly Tyr Met Phe Gly Lys Ala Ile Val

690 695 700

Cys Ser Asp Ala Ala Ala Glu Ala Ala Leu Tyr Gly Phe Thr Ala Val

705 710 715 720

Asp Arg Pro Glu Gly Tyr Leu Val Leu Ala Val Ala Ser Leu Gly Lys

725 730 735

Glu Ile Gln Glu Ile Thr Gly Thr Pro Gly Ser Glu Asp Val Lys Arg

740 745 750

Met Glu Glu Lys Lys Met Gly Val Lys Gly Val Gly Arg Lys Thr Thr

755 760 765

Asp Pro Ser Glu His Phe Thr Trp Arg Asp Gly Val Thr Val Pro Cys

770 775 780

Gly Lys Leu Val Pro Ser Thr Asn Lys Asp Gly Pro Leu Glu Tyr Asn

785 790 795 800

Glu Tyr Ala Val Tyr Asp Pro Lys Gln Val Ser Ile Ala Phe Leu Val

805 810 815

Gly Val Lys Tyr Glu Glu Gln Asn Met Glu Val Val Pro Asp Glu

820 825 830

Claims

1. The application of poly ADP-ribose polymerase gene in regulating drought tolerance of rice, wherein the coding amino acid sequence of the poly ADP-ribose polymerase gene is poly ADP-ribose polymerase shown as SEQ ID No. 2.

2. The use of claim 1, wherein the use of poly ADP-ribose polymerase gene for over-expression is used to enhance drought tolerance in rice.

3. The use according to claim 1, wherein the nucleotide sequence of the poly ADP-ribose polymerase gene is shown in SEQ ID No. 1.