CN114317559B - Rice salt stress-resistant gene mutant and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a rice salt stress tolerance gene mutant and application thereof, wherein the nucleotide sequence of the mutant is shown as SEQ ID NO. 1. The invention discovers the application of the OsCAMTA1 mutant in rice drought tolerance; cloning the gene and constructing a recombinant vector for the expression of the gene; transferring the vector into rice varieties Qinglin 157 and Ji Kedao 518 to obtain offspring transgenic rice plants and rice seeds; through the verification of the rice over-expressing the OsCAMTA1 mutant gene, the drought tolerance of the rice in the seedling stage is obviously improved, thereby proving that the rice over-expressing the gene obviously improves the drought tolerance of the rice in the seedling stage.

Description

Rice salt stress-resistant gene mutant and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a rice salt stress-resistant gene mutant and application thereof.

Background

Rice is affected for many periods due to drought conditions. Drought in the seedling stage of rice affects rice growth and even death. The improvement of drought tolerance of rice by genetic improvement is one of effective ways for improving the planting range and yield of rice. However, the drought response genes of plants are more than 1000, and the selection of which type or gene is used for drought resistance improvement of crops is always a difficult problem which puzzles the scientific community.

The drought-enduring major QTL (quantitative trait loci) utilized in breeding at present is mainly two loci of qSKC-1 and Saltol on rice chromosome 5 and 12. However, from the current results, the gene expression effect that has been selected is not ideal for drought tolerance of rice.

Disclosure of Invention

The invention aims to provide a rice salt stress-resistant gene mutant and application thereof, which can obviously improve drought tolerance of rice in seedling stage.

The first aspect of the invention provides a rice salt stress tolerance gene (OsCAMTA 1) mutant, the nucleotide sequence of which is shown as SEQ ID NO.1, and the amino acid sequence obtained by encoding the mutant is shown as SEQ ID NO. 2.

The second aspect of the present invention provides a vector carrying the above rice salt stress tolerance gene mutant.

Further, the vector is pCsV1300.

Further, the rice salt stress tolerance gene mutant is inserted between XbaI and BamHI in the multiple cloning site of pCsV1300.

The third aspect of the invention provides engineering bacteria containing the vector.

Furthermore, the engineering bacteria are escherichia coli.

The fourth aspect of the invention provides application of the rice salt stress tolerance gene mutant in improving rice drought tolerance.

Through research on rice calmodulin binding protein transcription factor family genes, the expression of the OsCAMTA1 gene is found to be obviously increased in drought environment; further, an OsCAMTA1 gene inactivation mutant is identified from a CRISPR/Cas9 rice mutant library (Oryza sativa L.var Nipponbare background), and the mutant is found to have reduced drought tolerance, so that the OsCAMTA1 plays a role in up-regulation in rice drought. In database Phytozome 12 (https:// Phytozome. Jgi. Doe. Gov/pz/portal. Html), the OsCAMTA1 gene (LOC_Os01g 69910) was found.

Further, according to the CDS sequence of OsCAMTA1 in the database, the pair of specific primers for amplifying full-length CDS are as follows:

OsCAMTA1-XbaI-F：5'-cgctctagaATGGCGGAGGGGCGGC-3'

OsCAMTA1-BamHI-R：5'-tgcggatccCTAGAAATATCCAGGCGTTGGC-3'。

PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 1min, annealing at 58℃for 30s, and extension at 72℃for 1min; the steps are circulated for 32 times; extending for 10min at 72 ℃.

The Open Reading Frame (ORF) nucleotide sequence of the OsCAMTA1 gene is 2637bp in length and encodes a protein consisting of 878 amino acids. The 1016bp site of the ORF is modified, namely G is mutated into A, so that the 339 rd arginine (Arg) of the encoded protein is changed into histidine (His), and the function of the protein on drought tolerance of rice is improved.

Further, the mutation process was amplified using the primers OsCAMTA1-XbaI-F/OsCAMTA 1-BamHI-R; the specific primer pairs are as follows:

1016-mu-F：5'-GCAGGAGACTTCCTTCATG-3'；

1016-mu-R：5'-GGGATCATGAAGGAAGTCTC-3'。

PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 1min, annealing at 58℃for 30s and extension at 72℃for 40s, the above steps were cycled 30 times; extending for 10min at 72 ℃.

Further, the rice salt stress tolerance gene mutant is used for promoting the expression of an ABA signal pathway gene in rice, wherein the ABA signal pathway gene is OsbZIP72 (LOC_Os09 g 28310), osDREB2A (LOC_Os01g07120), osDREB1A (LOC_Os09 g 35030) and OsPM1 (LOC_Os05g31670).

The fifth aspect of the invention provides a drought-resistant recombinant cell of rice, which is coated with a salt-tolerant stress gene mutant of rice or the vector.

The sixth aspect of the invention provides a construction method of the drought-resistant recombinant cell of rice, comprising the following steps: and infecting the rice callus cells by using the agrobacterium containing the vector to obtain the rice drought-resistant recombinant cells.

Further, the rice is Qinglin 157 or Ji Kedao 518.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the invention discovers the application of OsCAMTA1 in rice drought tolerance;

cloning the gene and constructing a recombinant vector for the expression of the gene;

transferring the vector into rice varieties Qinglin 157 and Ji Kedao 518 to obtain offspring transgenic rice plants and rice seeds; through the real verification of the rice over-expressing the OsCAMTA1 gene, the drought tolerance of the rice in the seedling stage is obviously improved, so that the rice seedling stage drought tolerance is obviously improved through the over-expression of the gene;

the drought response gene expression of the ABA signal pathway of the transgenic rice is obviously improved under drought conditions; further proves that the drought tolerance of the rice is improved by enhancing the ABA response path after the gene is over-expressed;

the rice over-expressed with the OsCAMTA1 gene has obviously improved drought tolerance, and can be used for transgenic breeding.

Drawings

FIG. 1 is a schematic diagram showing the structure of the overexpression vector pCsV1300 in example 2.

FIG. 2 is a schematic representation of the expression level of transgenic rice OsCAMTA1 in example 4.

FIG. 3 is a schematic representation of survival of transgenic plants overexpressing Qinglin 157, ji Kedao 518 and OsCAMTA1 in example 5.

FIG. 4 is a schematic representation of the dry weight of transgenic plants overexpressing Qinglin 157, ji Kedao 518 and OsCAMTA1 in example 5.

FIG. 5 is a schematic diagram showing the expression level of a gene involved in drought tolerance in rice in OsCAMTA1 transgenic rice in example 5.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

Example 1

Cloning and mutation of rice salt stress-tolerant gene OsCAMTA1

1. Extraction of RNA

Total RNA from rice leaves was extracted using TRIzol reagent (Invitrogen, USA).

(1) Taking leaves of Qinglin 157 of a rice variety growing to a three-leaf one-heart period, putting the leaves into a 2mL tube filled with 2 steel balls, and placing the leaves into liquid nitrogen;

(2) Grinding samples by using a tissue breaker, putting an adapter required by grinding samples into liquid nitrogen in advance for precooling, putting a tube filled with samples into the adapter, putting the tube on a machine, grinding the samples to powder at the frequency of 1400rpm/s for 90 s;

(3) Adding 1mL TRIzol reagent extracting solution into the sample tube, and rapidly and uniformly mixing the sample and the extracting solution by using a vortex instrument;

(4) Placing the lysate at 15-25deg.C for 10min to ensure that the sample is sufficiently lysed, and sucking out magnetic beads with magnet;

(5) Transferring to a desk type high-speed centrifuge, and centrifuging at 12,000Xg at 4deg.C for 10min;

(6) The supernatant was aspirated into a fresh 1.5mL tube, and 0.2mL of chloroform was added to each sample. Covering the cover, and fully vibrating each sample for 30s by using a vortex instrument, and standing for 2-15min at 15-25 ℃;

(7) Centrifuging at 2-8deg.C for 15min at 12,000Xg;

(8) After centrifugation, 3 layers are generated, and the colorless liquid at the uppermost layer is transferred into a new centrifuge tube;

(9) Adding 500mL of isopropanol into each sample, reversing the mixture upside down, and fully and uniformly mixing the mixture; standing at 15-25deg.C for 5-10min to allow RNA to precipitate completely;

(10) Centrifuging at 2-8deg.C for 10min at 12,000Xg, and removing supernatant;

(11) Adding 1mL of 75% ethanol prepared by DEPC (diethyl pyrocarbonate) water, fully rinsing RNA precipitate by upside down, and removing the supernatant;

(12) Centrifuging at 2-8deg.C for 5min at 7500 Xg, and removing supernatant;

(13) Centrifuging at 2-8deg.C for 7500 Xg for a short time, sucking away excessive ethanol with a gun, and drying in air for 5-10min (not too dry or not easily dissolved);

(14) Adding 30 mu L of DEPC treatment water, and fully dissolving RNA;

(15) The concentration of each sample was determined using a NanoDrop2000, and an A260/A280 value of 2.0-2.2 was acceptable. mu.L of RNA was taken and detected by agarose gel electrophoresis. The sample is stored in an ultralow temperature refrigerator at the temperature of-80 ℃ for standby.

2. cDNA Synthesis

4. Mu.g of RNA was reverse transcribed, and the RNA was reverse transcribed into cDNA using a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit (TransGen Biotech, china). The cDNA synthesis reaction system is shown in Table 1 below:

TABLE 1 cDNA Synthesis reaction System

The above liquids are mixed uniformly, for example, jian Lixin. The reaction condition is 42 ℃ for 30min; the reaction was terminated at 85℃for 5 seconds, and the cDNA concentration was measured using a NanoDrop 2000.

3. Full-length acquisition of OsCAMTA1 Gene coding region (CDS)

According to the CDS sequence of OsCAMTA1 in a database, a pair of specific primers (OsCAMTA 14-F/OsCAMTA 1-R) capable of amplifying full-length CDS are designed by utilizing Primer5.0 software, and a restriction enzyme cutting site is added at the 5' end of the primers for facilitating the construction of a next vector, so that a pair of primers (OsCAMTA 1-XbaI-F/OsCAMTA 1-BamHI-R) used for obtaining a rice salt stress tolerance gene OsCAMTA1 through the next PCR amplification is obtained. The full length of CDS of the expected size was successfully obtained using the reverse transcribed cDNA as a template. PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 1min, annealing at 58℃for 30s, and extension at 72℃for 1min; the steps are circulated for 32 times; extending for 10min at 72 ℃.

OsCAMTA1-XbaI-F：5'-cgctctagaATGGCGGAGGGGCGGC-3'

OsCAMTA1-BamHI-R：5'-tgcggatccCTAGAAATATCCAGGCGTTGGC-3'

After the reaction is finished, electrophoresis is carried out, products are recovered, the recovered fragments are connected into a vector pMD18-T, escherichia coli is transformed, simple clones are selected for sequencing, and CDS full length with complete reading frame, no mismatch and no frame shift is obtained.

4. Point mutation of OsCAMTA1 CDS

The 1016bp site G of the CDS was subjected to point mutation, and mutation primers 1016-mu-F and 1016-mu-R were designed. PCR was performed 2 times with cloned OsCAMTA1 CDS as a template (OsCAMTA 1-XbaI-F/1016-mu-R;1016-mu-F/OsCAMTA 1-BamHI-R), followed by a PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 1min, annealing at 58℃for 30s and extension at 72℃for 40s, the above steps were cycled 30 times; extending for 10min at 72 ℃. The mixture after dilution of the 2 PCR amplification products was used as a template for amplification using the primers OsCAMTA1-XbaI-F/OsCAMTA 1-BamHI-R.

1016-mu-F：5'-GCAGGAGACTTCCTTCATG-3'

1016-mu-R：5'-GGGATCATGAAGGAAGTCTC-3'

After the reaction is finished, electrophoresis is carried out, products are recovered, the recovered fragments are connected into a vector pMD18-T, escherichia coli is transformed, jian Kelong is picked up and sequenced, and the mutated OsCAMTA1 CDS sequence is obtained.

The nucleotide sequence is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2.

Example 2

Construction of OsCAMTA1 mutant over-expression vector

(1) Double-enzyme cutting binary vector pCsV1300 by XbaI and BamHI, and running gel to recover large fragment (vector);

(2) The T vector containing OsCAMTA1 mutant obtained in practical example 1 was digested with XbaI and BamHI, and then digested and gummed to recover CDS fragment containing OsCAMTA1 mutant;

(3) Ligating the recovered vector with the gene;

(4) E, converting escherichia coli competence, and selecting simple clones for PCR detection;

(5) For the simple clone detected positive overnight culture, plasmid was extracted for enzyme digestion verification.

Example 3

Agrobacterium-mediated rice genetic transformation system and identification (see in particular: cui Ying, cai Chaoxia, lin Yongjun, chen Hao. (2018.) Agrobacterium-mediated rice rapid transformation. Bio-101: e 1010176.)

(1) Selecting mature and plump rice varieties Qinglin 157 and Ji Kedao 518, and removing hulls; sterilizing with 75% alcohol for 1-2min, and pouring out alcohol; washing with sterilized distilled water for 2 times; soaking in 0.15% mercuric chloride (containing 0.1% tween 20) for 15-18min, and shaking for several times; the mercuric chloride was removed and rinsed 5 times with sterile distilled water. Inoculating the sterilized seeds into an induced callus culture medium, and culturing for 5-10d at 32 ℃ under illumination;

(2) Transforming the expression vector containing the OsCAMTA1 mutant obtained in example 2 into agrobacterium; in the first 2d of infection, streaking agrobacterium on LB medium containing 50mg/L kanamycin, and culturing at 28 ℃;

(3) Before infection, scraping activated agrobacterium into a suspension culture medium, performing shake culture at 28 ℃ and 180rpm for 3-3.5 hours, and then adjusting the concentration of bacterial liquid to be OD600 = 0.1-0.2 by using the suspension culture medium; placing the induced callus for 5-10d into agrobacterium suspension, and infecting for 1.5-10min; pouring out the bacterial liquid, and sucking the bacterial liquid on the surface of the callus by using sterilizing filter paper; the wound surface is covered with sterilized filter paper, and the wound is dried for 30min by a super clean bench. After blow drying, transferring the callus into a co-culture medium with a layer of sterilizing filter paper covered on the surface, performing dark culture at 20 ℃ for overnight, and transferring into a 25 ℃ incubator for continuous dark culture for 2d;

(4) After co-cultivation, the calli were transferred to an empty sterile container with forceps. Repeatedly cleaning the callus with sterilized distilled water for 7-8 times, wherein the first 3 times can be rapidly cleaned, and the second 3-4 times can be respectively soaked for 3-5min. Finally, the calli were soaked with sterilized distilled water containing 500mg/L Carbenicillin (Cn) for 30min. Pouring Cn solution, sucking water on the surface of the callus with sterilized filter paper as much as possible, covering the surface of the callus with a layer of sterilized filter paper, and drying with a clean bench for 1h;

(5) Placing the cleaned calli on a screening culture medium containing hygromycin at 32 ℃ and culturing for 14d under illumination;

(6) After 14d of screening, the resistant calli were transferred to differentiation medium and incubated at 28 ℃ (photoperiod 14h light/10 h dark);

(7) When the resistant callus forms 3-4cm high regenerated seedlings on the differentiation medium, transferring the regenerated seedlings into a rooting medium for culture until complete transgenic rice plants are formed. The inbred offspring of transgenic rice can be screened by hygromycin for homozygous transgenic plants.

3 different transgenic lines were selected from transgenic rice of qinglin 157, ji Kedao 518, namely: osCAMTA1OX-1, osCAMTA1OX-2 and OsCAMTA1OX-3.

Example 4

Detection of expression level of OsCAMTA1 mutant in T2 generation homozygous transgenic rice

Leaves of Wild Type (WT) rice and transgenic rice plants grown to the trefoil-heart stage were taken and RNA was extracted for analysis of the relative expression amount of genes. RT-qPCR was performed using Real-time PCR apparatus (ABI, USA). RNA extraction and cDNA Synthesis were performed as in example 1. The cDNA was then diluted 10-fold and RT-qPCR was performed as described in kit THUNDERBIRD SYBR qPCR Mix Without Rox (TOYOBO, japan). Primers used for RT-qPCR were designed by Primer Express 3.0, osGAPDH was used as an internal reference gene, and the primers used were as follows:

qRT-OsCAMTA1-XbaI-F：5'-cgctctagaATGGCGGAGGGGCGGC-3'；

qRT-OsCAMTA1-BamHI-R：5'-tgcggatccCTAGAAATATCCAGGCGTTGGC-3'；

OsGAPDH-F：5'-ctgagaataaaacgtggacggtg-3'；

OsGAPDH-R：5'-tccatatcatcagcatcgttacaac-3'。

reaction conditions for RT-qPCR: pre-denaturation at 95℃for 10min; denaturation at 95℃for 15sec, annealing at 60℃for 1min, 40 cycles of this step.

Calculating according to the formula 2-delta Ct; wherein DeltaCt= (Ct target gene-Ct reference gene), ΔΔct= (Δctsample- Δct control), ct is fluorescence threshold.

The results showed (as shown in FIG. 2) that the expression level of the OsCAMTA1 mutant in the OsCAMTA1 mutant overexpressing plant was increased by about 60-fold or more as compared with the control.

Example 5

Drought tolerance test of OsCAMTA1 mutant over-expression plants

Transgenic plants overexpressing Qinglin 157, ji Kedao 518 and homozygous OsCAMTA1 mutants grown to trefoil-heart stage were transferred to drought environments, respectively, and after 5d treatment phenotypes and statistical survival were observed and dry weight recorded.

The results show that compared with wild-type qinglin 157 and wild-type Ji Kedao 518, the tolerance of oscarma 1 mutant over-expression plants to drought is enhanced, and the survival rate (figure 3) and dry weight (figure 4) of the oscarma 1 mutant over-expression plants are obviously higher than those of wild-type qinglin 157 and wild-type Ji Kedao 518 varieties.

Analysis of the expression of drought tolerance-associated genes in rice shows that the expression of ABA signal pathway genes OsbZIP72 (LOC_Os09 g 28310), osDREB2A (LOC_Os01g 07120), osDREB1A (LOC_Os09 g 35030) and OsPM1 (LOC_Os05g31670) in the OsCAMTA1 mutant transgenic rice is remarkably increased.

Further, when RT-qPCR is performed on RNA, a primer pair is designed to take OsGAPDH as an internal reference gene, and the specific steps are as follows:

qRT-OsCAMTA1-XbaI-F：5'-cgctctagaATGGCGGAGGGGCGGC-3'；

qRT-OsCAMTA1-BamHI-R：5'-tgcggatccCTAGAAATATCCAGGCGTTGGC-3'；

OsGAPDH-F：5'-ctgagaataaaacgtggacggtg-3'；

OsGAPDH-R：5'-tccatatcatcagcatcgttacaac-3'。

reaction conditions for RT-qPCR: pre-denaturation at 95℃for 10min; denaturation at 95℃for 15sec, annealing at 60℃for 1min, 40 cycles of this step.

In conclusion, the gene OsCAMTA1 which is important to obtain color in the drought response process is obtained by early gene expression analysis and drought tolerance screening of a rice mutant library. The transgenic plant of homozygous T2 generation is obtained by successfully transforming the rice varieties Qinglin 157 and Ji Kedao 518 with the OsCAMTA1 mutant over-expression vector through an agrobacterium-mediated transformation method. Experiments prove that under drought conditions, the survival rate of the OsCAMTA1 mutant over-expression plants OsCAMTA1OX-1, osCAMTA1OX-2 and OsCAMTA1OX-3 is remarkably higher than that of Yu Qinglin, 157 and Ji Kedao. The above results indicate that the gene OsCAMTA1 mutant in rice can improve drought tolerance of rice.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

SEQUENCE LISTING

<110> Jilin agricultural sciences institute, shenzhen Huachun Chunhui technology Co., ltd

<120> a rice salt stress-tolerant gene mutant and application thereof

<130> 2.14

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 2637

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<213> (Synthesis)

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atgcagcacc agcaaggatt tgatacacac aggctacacc aggaagtaaa atcgcggtgg 60

ttaaagccta aagaggttct gcagatattg cagaatcatg atcggttcat catcacacat 120

aagactcctc ataagccacc gagtggtgct tggtttcttt tcaaccgtag agttcttcgg 180

tacttcagaa atgatgggta tgaatggcga aaaaagaaga atgggaagac tattgctgaa 240

gcccatgagc gccttaaggt tgataatgtt gatgcactga attgctacta tgcacacgca 300

gacaaaaact ctacatttca gaggcgtata tattggatgc ttgatccggc ttatgatcac 360

atagtttttg tccactacag agatgttcaa gagggcagca tttcagtatc agcactgaat 420

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ggattgacta gcgagctatt tgccccatgt cttaattcat gcagtccagg atctgcggaa 540

gaagttagtt cccagattat ggccataaac aacgaaacaa atagcgttag tcaacctgat 600

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gtgtatggga ttcagaatga agaaccaggc acttgtagaa atcttgcaga tgtttttagt 780

ggactggagt tcagcaaaga aaatcatcct gaagaaaccg gactcccttt ttccagcact 840

attgatgtgc tgaaaaattc agacacttgg ttggaggagg atcagattga agcaattctg 900

cattcagcat ctatgatagt gactgaaaac caatggttca atattcgtga ggtttcccca 960

gaatggtcct attgttctga aagtacaaag gttatcattg caggagactt ccttcatgat 1020

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cagcaaggtg tcattcgttg ccatactcca tgccttgatg ctagaaaagt gacaatgtac 1140

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cctaccaaaa gtgtggtttg tgagaacagg aaaccctgca gagaagtaca tgaatctgaa 1260

ctccatcaga ggcctactga aagcaacaat gagctattgt tgctttttaa ctatgctcag 1320

ttgcttttcg atggtcatgt ttctgagcaa tttttaaagt ttggcttgcc attcccaaat 1380

cttgaatgcg gactccaagt tagcccatct gaaattatga agggggcatc tgagcggctg 1440

aaccgtgaca ccgcagtaaa ttgtgtcatg gaagtactgc ttaacaacaa gtttgaagag 1500

tggctgtttt ctaaatatga acaaaatagt gaagggaatc attttctccc taggcaatac 1560

catggtgtta tacatacaat tgctgccttg gggtacaact gggctttgaa acttctgctt 1620

aattcaggtg tacttgtaaa ctaccgtgat gcaaatggat ggactgcttt acattgggct 1680

gcacgatttg gcagggaaga gacggtagtg ctccttcttg atgcaggtgc cgcagctggt 1740

gcactgtcag acccaacagc acaagatcct gctgccaaaa cacctgcttc agtagcttct 1800

gcatatggtt tcaagggcct ttctgcgtat ctttcagaag cagaactaat agctcatctt 1860

cattctctcg aatcaaaaga aaacgggtct tctggagatc aaatatcaag agttgtgggc 1920

agaatatcag acacatctgc ccatgcacaa agtggtagcg atgatcagct tgcactgaag 1980

gaatccctag gagctatgcg atatgctgtt caagctgccg ggcgcataca aactgccttc 2040

cgtatttttt ccttcaggaa gaaacaacaa gcaggtcttc agaatagagg taaccacatt 2100

atatccattc gtgaggtagg tgctgcttca cacggtatgc ttgagaaagc cgcgttatct 2160

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gtcatcaaga tacaggcgcg tgtcagagct catcaacaac ataacaagta caaggagtta 2280

cttcgaagtg tgggcatcct tgagaaggtc atgcttagat ggtaccgaaa gggtgttggt 2340

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cacaaacaaa acaaggatga tgatgaaaaa gtagaagttt ctccagcatc tcatgtttat 2580

ggcagtggca gtcatcacat gtgttggcta agccacaaca acaaagcaat gcactag 2637

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Lys Thr Pro Ala Ser Val Ala Ser Ala Tyr Gly Phe Lys Gly Leu Ser

595 600 605

Ala Tyr Leu Ser Glu Ala Glu Leu Ile Ala His Leu His Ser Leu Glu

610 615 620

Ser Lys Glu Asn Gly Ser Ser Gly Asp Gln Ile Ser Arg Val Val Gly

625 630 635 640

Arg Ile Ser Asp Thr Ser Ala His Ala Gln Ser Gly Ser Asp Asp Gln

645 650 655

Leu Ala Leu Lys Glu Ser Leu Gly Ala Met Arg Tyr Ala Val Gln Ala

660 665 670

Ala Gly Arg Ile Gln Thr Ala Phe Arg Ile Phe Ser Phe Arg Lys Lys

675 680 685

Gln Gln Ala Gly Leu Gln Asn Arg Gly Asn His Ile Ile Ser Ile Arg

690 695 700

Glu Val Gly Ala Ala Ser His Gly Met Leu Glu Lys Ala Ala Leu Ser

705 710 715 720

Ile Gln Lys Asn Phe Arg Cys Trp Lys Lys Arg Lys Glu Phe Leu Lys

725 730 735

Ile Arg Lys Asn Val Ile Lys Ile Gln Ala Arg Val Arg Ala His Gln

740 745 750

Gln His Asn Lys Tyr Lys Glu Leu Leu Arg Ser Val Gly Ile Leu Glu

755 760 765

Lys Val Met Leu Arg Trp Tyr Arg Lys Gly Val Gly Leu Arg Gly Phe

770 775 780

His Pro Gly Ala Ile Ala Met Pro Ile Asp Glu Glu Asp Glu Asp Asp

785 790 795 800

Val Ala Lys Val Phe Arg Lys Gln Arg Val Glu Thr Ala Leu Asn Lys

805 810 815

Ala Val Ser Arg Val Ser Ser Ile Ile Asp Ser Pro Val Ala Arg Gln

820 825 830

Gln Tyr Arg Arg Met Leu Lys Met His Lys Gln Asn Lys Asp Asp Asp

835 840 845

Glu Lys Val Glu Val Ser Pro Ala Ser His Val Tyr Gly Ser Gly Ser

850 855 860

His His Met Cys Trp Leu Ser His Asn Asn Lys Ala Met His

865 870 875

Claims (9)

1. A rice salt stress-resistant gene mutant is characterized in that the nucleotide sequence of the mutant is shown as SEQ ID NO. 1.

2. A vector carrying the rice salt stress tolerance gene mutant of claim 1.

3. The vector of claim 2, wherein the vector is pCsV1300.

4. The vector of claim 3, wherein the rice salt stress tolerance gene mutant is inserted between XbaI and BamHI in the multicloning site of pCsV1300.

5. An engineered bacterium comprising the vector of any one of claims 2-4.

6. The use of the salt stress-tolerant rice gene mutant according to claim 1 for improving drought tolerance of rice, wherein the salt stress-tolerant rice gene mutant is overexpressed.

7. The use according to claim 6, wherein the rice salt stress tolerance gene mutant is used to promote expression of ABA signaling pathway genes in rice, the ABA signaling pathway genes being OsbZIP72, osDREB2A, osDREB a and OsPM1.

8. The construction method of the drought-resistant recombinant cell of the rice is characterized by comprising the following steps: infecting rice callus cells with agrobacterium containing the vector of any of claims 2-4 to obtain the drought resistant recombinant rice cells.

9. The method of claim 8, wherein the rice is Qinglin 157 or Ji Kedao 518.