CN114317559A - Rice salt-tolerant stress 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 resistant gene mutant and application thereof, wherein the nucleotide sequence of the mutant is shown as SEQ ID No. 1. The invention finds the application of the OsCAMTA1 mutant in drought tolerance of rice; cloning the gene and constructing a recombinant vector for expression of the gene; transferring the vector into rice varieties Qinglin 157 and Jike rice 518 to obtain progeny transgenic rice plants and rice seeds; experiments prove that the drought tolerance of rice at the seedling stage is obviously improved by rice over-expressing the OsCAMTA1 mutant gene, so that the drought tolerance of the rice at the seedling stage is obviously improved by over-expressing the gene.

Description

Rice salt-tolerant stress 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

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

At present, drought-enduring major QTL (quantitative trait loci) utilized in breeding are mainly two sites of qSKC-1 and Saltol on No. 5 and No. 12 chromosomes of rice. However, from the results of the present, the gene expression effect that has been selected is not ideal for drought tolerance in rice.

Disclosure of Invention

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

The first aspect of the invention provides a rice salt-tolerant stress gene (OsCAMTA1) mutant, wherein the nucleotide sequence of the mutant is shown as SEQ ID NO.1, and the amino acid sequence obtained by coding the mutant is shown as SEQ ID NO. 2.

The second aspect of the invention provides a carrier carrying the rice salt-tolerant stress gene mutant.

Further, the vector is pCsV 1300.

Furthermore, the multiple cloning site of the rice salt-tolerant stress gene mutant inserted into the pCsV1300 is between XbaI and BamHI.

The third aspect of the invention provides an engineering bacterium containing the carrier.

Further, the engineering bacteria is escherichia coli.

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

Through research on rice calmodulin binding protein transcription factor family genes, the OsCAMTA1 gene expression is found to be remarkably increased in a drought environment; an OsCAMTA1 gene inactivation mutant is further identified from a CRISPR/Cas9 rice mutant library (Oryza sativa L. var Nipponbare background), and the mutant is found to have reduced drought tolerance, which indicates that OsCAMTA1 plays a positive regulation role in rice drought. The OsCAMTA1 gene (LOC _ Os01g69910) was found in the database Phytozome 12(https:// Phytozome. jgi. doe. gov/pz/portal. html).

Further, based on the CDS sequence of oscalmta 1 in the database, a pair of specific primers for amplification of full-length CDS is as follows:

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

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

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

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

Further, the mutation process was amplified using primer 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 5 min; denaturation at 94 ℃ for 1min, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 40s, wherein the steps are circulated for 30 times; stretching for 10min at 72 ℃.

Further, the rice salt-tolerant stress gene mutant is used for promoting expression of ABA signal pathway genes in rice, wherein the ABA signal pathway genes are OsbZIP72(LOC _ Os09g28310), OsDREB2A (LOC _ Os01g07120), OsDREB1A (LOC _ Os09g35030) and OsPM1(LOC _ Os05g 31670).

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

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

Further, the rice is Qinglin 157 or Jike rice 518.

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

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

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

transferring the vector into rice varieties Qinglin 157 and Jike rice 518 to obtain progeny transgenic rice plants and rice seeds; experiments prove that the drought tolerance of rice at the seedling stage is obviously improved by rice over-expressing the OsCAMTA1 gene, so that the drought tolerance of the rice at the seedling stage is obviously improved by over-expressing the gene;

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

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

Drawings

FIG. 1 is a schematic structural view of an overexpression vector pCsV1300 in example 2.

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

FIG. 3 is a schematic representation of the survival rates of transgenic plants overexpressing Genglin 157, Jike rice 518 and OsCAMTA1 in example 5.

FIG. 4 is a schematic representation of the dry weight of transgenic plants overexpressing Genglin 157, Jike rice 518 and OsCAMTA1 in example 5.

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

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

Cloning and mutation of rice salt-tolerant stress gene OsCAMTA1

1. Extraction of RNA

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

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

(2) grinding a sample by using a tissue crusher, pre-cooling an adapter required by the sample grinding in liquid nitrogen in advance, loading a pipe filled with the sample into the adapter, loading the pipe on a machine, wherein the frequency is 1400rpm/s, the sample grinding time is generally 90s, and grinding the sample into powder;

(3) adding 1mL of 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 15-25 deg.C for 10min to ensure that the sample is fully lysed, and simultaneously sucking out the magnetic beads with a magnet;

(5) transferring to a desk type high speed centrifuge, and centrifuging at 12,000 Xg and 4 ℃ for 10 min;

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

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

(8) 3 layers can be generated after centrifugation, and the colorless liquid at the uppermost layer is transferred to a new centrifugal tube;

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

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

(11) adding 1mL of 75% ethanol prepared by DEPC (diethyl pyrocarbonate) water, fully rinsing RNA precipitate by reversing the mixture from top to bottom, and removing supernatant;

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

(13) centrifuging at 7500 Xg for a short time at 2-8 deg.C, sucking off excessive ethanol with a gun, and drying in air for 5-10min (not too dry, otherwise not easy to dissolve);

(14) adding 30 mu L of DEPC treated water to fully dissolve RNA;

(15) the concentration of each sample was measured by using NanoDrop2000, and the A260/A280 value was found to be acceptable at 2.0-2.2. 2. mu.L of RNA was subjected to agarose gel electrophoresis for detection. The samples were stored in an ultra low temperature freezer at-80 ℃ for future use.

2. Synthesis of cDNA

Mu.g of RNA was reverse transcribed and the RNA was reverse transcribed into cDNA using 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

Mixing the above liquids, and centrifuging. The reaction condition is 42 ℃ and 30 min; the reaction was stopped at 85 ℃ for 5s and the cDNA concentration was determined using NanoDrop 2000.

3. Obtaining the full Length of coding region (CDS) of OsCAMTA1 Gene

According to the CDS sequence of OsCAMTA1 in a database, a pair of specific primers (OsCAMTA14-F/OsCAMTA1-R) capable of amplifying the full-length CDS is designed by using Primer5.0 software, and a pair of primers (OsCAMTA1-XbaI-F/OsCAMTA1-BamHI-R) used for obtaining a rice salt-tolerant stress gene OsCAMTA1 by the next PCR amplification is obtained by adding restriction enzyme cutting sites at the 5' ends of the primers for facilitating the construction of a next vector. The CDS full length of the expected size was successfully obtained using the reverse transcribed cDNA as a template. PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 1min, annealing at 58 deg.C for 30s, and extension at 72 deg.C for 1 min; the steps are circulated for 32 times; stretching 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 picked for sequencing, and the CDS full length with a complete reading frame, no mismatching and no frame shifting is obtained.

4. Point mutation of OsCAMTA1 CDS

The 1016bp site G of CDS was subjected to point mutation to design mutation primers 1016-mu-F and 1016-mu-R. Using the cloned OsCAMTA1 CDS as a template, 2 PCR amplifications were performed (OsCAMTA 1-XbaI-F/1016-mu-R; 1016-mu-F/OsCAMTA1-BamHI-R), PCR program: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 40s, wherein the steps are circulated for 30 times; stretching for 10min at 72 ℃. Using the mixture obtained after dilution of the 2 PCR amplification products as a template, amplification was carried out using the primer OsCAMTA1-XbaI-F/OsCAMTA 1-BamHI-R.

1016-mu-F：5'-GCAGGAGACTTCCTTCATG-3'

1016-mu-R：5'-GGGATCATGAAGGAAGTCTC-3'

And after the reaction is finished, carrying out electrophoresis, recovering a product, connecting the recovered fragment into a vector pMD18-T, transforming escherichia coli, selecting simple clone for sequencing, and obtaining a mutant OsCAMTA1 CDS sequence.

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 overexpression vector

(1) The binary vector pCsV1300 is digested by XbaI and BamHI, and a large fragment (vector) is recovered by running gel;

(2) the T vector containing the OsCAMTA1 mutant obtained in example 1 was digested with XbaI and BamHI, and the CDS fragment containing the OsCAMTA1 mutant was recovered from the gel after digestion;

(3) connecting the recovered vector with a gene;

(4) transforming escherichia coli competence, and selecting simple clone for PCR detection;

(5) and (5) carrying out overnight culture on the simple clone which is detected to be positive, and extracting the plasmid for enzyme digestion verification.

Example 3

Agrobacterium-mediated genetic transformation system and identification of rice (see in particular: Traine, Cai, Lin champion, Chenhao. (2018). Agrobacterium-mediated Rapid transformation of rice Bio-101: e1010176.)

(1) Selecting mature and plump rice varieties of Qinglin 157 and Jike rice 518, and shelling; sterilizing with 75% ethanol for 1-2min, and removing ethanol; washing with sterilized distilled water for 2 times; adding 0.15% mercuric chloride (containing 0.1% Tween 20) and soaking for 15-18min, and shaking for several times; mercury mercuric oxide was poured off, and washed with sterilized distilled water 5 times. Inoculating the sterilized seeds into an induction callus culture medium, and culturing for 5-10 days at 32 ℃ under illumination;

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

(3) before infection, scraping the activated agrobacterium into a suspension culture medium, performing shake culture at 28 ℃ and 180rpm for 3-3.5h, and then adjusting the concentration of a bacterial liquid to OD600 of 0.1-0.2 by using the suspension culture medium; placing the callus for inducing for 5-10 days into the agrobacterium tumefaciens suspension, and infecting for 1.5-10 min; pouring out the bacterial liquid, and sucking the bacterial liquid on the callus surface by using sterilization filter paper; covering sterilized filter paper on the surface of the callus, and drying for 30min by an ultra-clean bench. After drying, transferring the callus into a co-culture medium with a layer of sterilized filter paper covered on the surface, performing dark culture at 20 ℃ for overnight, and then transferring into an incubator at 25 ℃ for continuous dark culture for 2 d;

(4) after the co-culture was completed, the callus was transferred to an empty sterilized container with forceps. Repeatedly cleaning with sterilized distilled water for 7-8 times, wherein the first 3 times can be rapidly cleaned, and the last 3-4 times can be soaked for 3-5min each time. Finally, the callus is soaked in sterilized distilled water containing 500mg/L Carbenicillin (Cn) for 30 min. Pouring the Cn solution, sucking water on the callus surface with sterile filter paper as much as possible, covering a layer of sterile filter paper on the callus surface, and drying for 1h by using an ultra-clean bench;

(5) placing the cleared callus on a screening culture medium containing hygromycin for 32 ℃, and culturing for 14d by illumination;

(6) after screening for 14 days, transferring the resistant callus into a differentiation culture medium, and culturing at 28 ℃ (photoperiod is 14h light/10 h dark);

(7) when the resistance callus forms a regeneration seedling with the height of 3-4cm on a differentiation culture medium, the regeneration seedling is transferred into a rooting culture medium for culture until a complete transgenic rice plant is formed. The inbred progeny of the transgenic rice can adopt hygromycin to screen homozygous transgenic plants.

From transgenic rice plants of Qinglin 157, Jike rice 518, 3 different transgenic lines were selected, namely: OsCAMTA1OX-1, OsCAMTA1OX-2 and OsCAMTA1 OX-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 growing to the three-leaf one-heart stage are taken, and RNA is extracted for analysis of relative gene expression. RT-qPCR was performed using a Real-time PCR instrument (ABI, USA). RNA extraction and cDNA Synthesis were performed as in example 1. The cDNA was then diluted 10-fold and subjected to RT-qPCR according to the kit THUNDERBIRD SYBR qPCR Mix Without Rox (TOYOBO, Japan). Primers for RT-qPCR were designed by Primer Express 3.0, with OsGAPDH as the 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 of RT-qPCR: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 1min, and this step is repeated for 40 cycles.

Calculating according to a formula 2-delta Ct; wherein, the delta Ct is equal to (Ct target gene-Ct internal reference gene), the delta Ct is equal to (delta Ct sample-delta Ct control), and Ct is a fluorescence threshold value.

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

Example 5

Drought tolerance test of OsCAMTA1 mutant overexpression plants

The transgenic plants overexpressing Genglin 157, Jike rice 518 and homozygous OsCAMTA1 mutant which grow to the three-leaf one-heart stage are respectively transferred to a drought environment, and after 5d of treatment, the phenotype and the survival rate are observed and counted, and the dry weight is recorded.

The results show that compared with the wild-type Qinglin 157 and the wild-type Jike rice 518, the OsCAMTA1 mutant over-expression plants have enhanced drought tolerance, and the survival rate (figure 3) and the dry weight (figure 4) of the over-expression plants are obviously higher than those of the wild-type Qinglin 157 and the wild-type Jike rice 518.

Expression analysis of drought-tolerant related genes in rice discovers that expression of ABA signal pathway genes OsbZIP72(LOC _ Os09g28310), OsDREB2A (LOC _ Os01g07120), OsDREB1A (LOC _ Os09g35030) and OsPM1(LOC _ Os05g31670) in OsCAMTA1 mutant transgenic rice is obviously improved.

Further, when RT-qPCR is performed on RNA, a primer pair is designed with OsGAPDH as an internal reference gene, specifically 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 of RT-qPCR: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 1min, and this step is repeated for 40 cycles.

In conclusion, the gene OsCAMTA1 which obtains important coloration in the drought response process is obtained by performing early gene expression analysis and drought tolerance screening on a rice mutant library. The OsCAMTA1 mutant overexpression vector is successfully transformed into rice varieties Genglin 157 and Jike rice 518 by an agrobacterium-mediated transformation method, and homozygous T2 generation transgenic plants are obtained. Experiments prove that under drought conditions, the survival rates of OsCAMTA1 mutant overexpression plants OsCAMTA1OX-1, OsCAMTA1OX-2 and OsCAMTA1OX-3 are obviously higher than those of Genglin 157 and Jikou rice 518. The results show that the OsCAMTA1 mutant gene in rice can improve the drought tolerance of rice.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

SEQUENCE LISTING

<110> Jilin agricultural science and technology institute, Shenzhen Huachunshui science and technology Limited

<120> salt stress resistant gene mutant of rice and application thereof

<130> 2.14

<160> 2

<170> PatentIn version 3.3

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ggactggagt tcagcaaaga aaatcatcct gaagaaaccg gactcccttt ttccagcact 840

attgatgtgc tgaaaaattc agacacttgg ttggaggagg atcagattga agcaattctg 900

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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

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cttcgaagtg tgggcatcct tgagaaggtc atgcttagat ggtaccgaaa gggtgttggt 2340

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Ala Arg Phe Gly Arg Glu Glu Thr Val Val Leu Leu Leu Asp Ala Gly

565 570 575

Ala Ala Ala Gly Ala Leu Ser Asp Pro Thr Ala Gln Asp Pro Ala Ala

580 585 590

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 (10)

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

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

3. The vector of claim 2, wherein the vector is pCsV 1300.

4. The vector of claim 3, wherein the rice salt stress tolerant gene mutant is inserted into the pCsV1300 at a multiple cloning site between XbaI and BamHI.

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

6. The use of the mutant of the rice salt-tolerant stress gene of claim 1 for improving drought tolerance of rice.

7. The use of claim 6, wherein the rice salt-tolerant stress gene mutant is used for promoting expression of ABA signal pathway genes in rice, and the ABA signal pathway genes are OsbZIP72, OsDREB2A, OsDREB1A and OsPM 1.

8. A drought-resistant recombinant cell of rice, which comprises the salt stress-resistant gene mutant of rice of claim 1 or the vector of any one of claims 2 to 4.

9. The method for constructing the drought resistant recombinant rice cell of claim 8, comprising the following steps: infecting rice callus cells with agrobacterium containing the vector of any one of claims 2-4 to obtain the drought resistant rice recombinant cells.

10. The method of claim 9, wherein the rice is Qinglin 157 or Jikoku 518.

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