CN114085854A

CN114085854A - Rice drought-resistant and salt-tolerant gene OsSKL2 and application thereof

Info

Publication number: CN114085854A
Application number: CN202111540270.8A
Authority: CN
Inventors: 武健东; 刘雨晴; 李爱琪; 蒋迎利; 章琴
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2021-12-16
Filing date: 2021-12-16
Publication date: 2022-02-25
Anticipated expiration: 2041-12-16
Also published as: CN114085854B

Abstract

The invention discloses a rice drought resistance and salt tolerance gene OsSKL2 and its application, and relates to the technical field of genetic engineering. The gene has the nucleotide sequence shown in SEQ ID NO.1. The gene can regulate the survival rate of rice under drought and high salinity, and has a positive regulatory effect on rice drought and high salinity. Biological adversity provides a new research approach, which has important theoretical and practical significance for cultivating drought-resistant and salt-tolerant materials.

Description

Rice drought-resistant and salt-tolerant gene OsSKL2 and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a drought-resistant and salt-tolerant rice gene OsSKL2 and application thereof.

Background

Rice is the main grain crop in the world, and provides a ration for more than half of the population all over the world. However, as population grows, environmental conditions deteriorate and the cultivated land area is reduced, the contradiction between population increase and food demand becomes more and more prominent. At present, the main environmental factors causing the decrease of rice yield are drought and soil salinization. According to statistics, the rice planting area and the yield depending on the traditional flooding irrigation mode account for about 55 percent and 75 percent of the total rice area and the total rice yield in the world. In recent years, the yield reduction of grains caused by drought and soil salinization is increased, and the safe production of grains is threatened. Therefore, the digging of drought-resistant salt-tolerant genes and the analysis of the action mechanism thereof are important targets of biotechnology breeding and have important significance for ensuring the high and stable yield of rice and the safe production of grains.

In order to adapt to the change of the external environment, plants have evolved various ways and regulation mechanisms to respond to environmental stimuli in the long-term evolution process so as to ensure normal growth and development. Research shows that after the stress receptors on the surface (such as cell wall, plasma membrane) and organelles (such as cytoplasm and nucleus) of plant cell capture external stress signals, the stress receptors can rapidly transmit the stimulation signals into the cell to influence a second signal (such as Ca)²⁺ROS, NO, phospholipids, etc.), thereby altering expression of regulatory genes and transcription factors. The transcription factor can ensure the normal growth of the plant by combining and regulating the expression of downstream functional genes to induce plant protection mechanisms such as detoxification and stress response repair. The corn transcription factor ZmbHLH124 is directly combined with and activates the expression of ZmDREB2A, thereby improving the drought tolerance of corn. In rice, OsMYB3R-2 is obtained by regulating OsCycB 1; 1 and OsKNOLLE2, reduces ABA sensitivity, and enhances the cold, high salt and drought resistance of rice. Although a plurality of genes for plant drought and salt tolerance have been reported, the primary metabolism and the secondary metabolism pathways directly participate in the drought and salt stressThe functional major genes of (A) are poorly understood.

The shikimic acid cycle is widely present in plants and microorganisms, is an important hub connecting carbohydrate metabolism and secondary metabolism, and provides substrates for other secondary metabolism. The shikimate pathway is a biochemical reaction catalyzed by a variety of metabolic enzymes, the products of which are phenylalanine, tyrosine and tryptophan. Wherein shikimic acid kinase (SK) catalyzes the fifth step reaction of shikimic acid pathway, and the coding genes of SK in rice are SK-1, SK-2, SK-3, SKL1 and SKL 2. The development of SK1 and SK2 and chloroplasts was found to be closely related. Although research shows that the content of shikimic acid is obviously changed under drought and high-salt conditions, no direct evidence exists on how to regulate the drought and high-salt stress reaction, and the functional gene is unclear. Therefore, the rice drought-resistant and salt-tolerant gene OsSKL2 and the application thereof are provided.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a drought-resistant and salt-tolerant rice gene OsSKL2 and application thereof.

The invention realizes the purpose through the following technical scheme:

the invention provides a drought-resistant and salt-tolerant gene OsSKL2 of rice, which has a nucleotide sequence shown as SEQID NO.1 and a full length of 1128 bp.

The invention also provides application of the rice drought-resistant and salt-tolerant gene OsSKL2 in improving the drought resistance and salt tolerance of rice.

The further improvement is that the gene OsSKL2 has positive regulation and control effects on drought resistance and salt tolerance of rice, the survival rate of rice under drought and high salt conditions can be improved by over-expressing transgenic rice plants, and the survival rate of RNAi silent transgenic rice plants under drought and high salt conditions is obviously reduced

The invention also provides a coding protein of the rice drought-resistant and salt-tolerant gene OsSKL2, and the coding protein has an amino acid sequence shown as SEQID NO. 2.

The further improvement is that the protein is located in chloroplast and is expressed under drought and high-salt induction, and the coded shikimate kinase is a key metabolic enzyme for catalyzing shikimate pathway.

The invention also provides a plasmid vector, which is obtained by inserting the rice drought-resistant and salt-tolerant gene OsSKL2 into a pEASY-T1 vector.

The invention also provides a genetically engineered host cell, which is the Escherichia coli competent Trans5 alpha cell of the plasmid vector.

The invention also provides a method for obtaining the rice drought-resistant and salt-tolerant gene OsSKL2, which comprises the steps of designing a specific amplification primer by taking the nucleotide sequence of the OsSKL2 gene as a template, taking the rice Nipponbare variety as a material, extracting RNA and carrying out reverse transcription to obtain cDNA, and obtaining the gene OsSKL2 by a PCR amplification technology.

The further improvement is that the specific amplification primers for PCR amplification are as follows:

SKL2–F-1：ATGAGGATAGGGGCAGGGGCGAACA；

SKL2–R-1：TCAGATATTGGTCGGTGGGGCGTCG。

the invention also provides an over-expression vector, which is a pCAMBIA1301 plant expression vector, wherein the multi-cloning site region of the over-expression vector is sequentially connected with a 35S promoter, the gene OsSKL2 and an NOS terminator.

The invention also provides a subcellular localization vector, which is obtained by connecting the OsSKL2 gene and the pCAMBIA1305 vector after being cut by SpeI and BamHI, in particular to pCAMBIA1305-SKL2 gene, and the upstream and downstream primers are used for connection as follows:

OsSKL2-F：ATGTTGGCCTCCACTTGCTTCTCCG；

OsSKL2-R：TATGTTGGTGGGTGGTGCGTCGGA。

the invention has the following beneficial effects:

compared with the prior art, the invention has the following advantages: the invention provides a drought-resistant and salt-tolerant gene OsSKL2 of rice and application thereof, wherein the gene can regulate the survival rate of rice under drought and high salt conditions, and has positive regulation and control effects on the drought and high salt conditions of rice; the discovery of the gene provides direct evidence for the regulation and control effect of shikimic acid under abiotic stress, provides a new research approach for improving abiotic stress of crops, and has important theoretical and practical significance for cultivating drought-resistant and salt-tolerant materials.

Drawings

FIG. 1 is a map of the RNAi silencing vector pEASY-T1;

FIG. 2 is a map of an over-expressed pCAMBIA1301 vector;

FIG. 3 is a high-salt, drought-induced expression pattern diagram of OsSKL 2; respectively treating wild rice middle flowers with the size of four weeks by using 100mM mannitol solution and 200mM NaCl solution for 11 hours, and then sampling and analyzing after 12 hours;

FIG. 4 is a map of OsSKL2 subcellular localization analysis;

FIG. 5 is a diagram of molecular characterization of OsSKL2 overexpression and RNAi silencing strains; WT is wild type, OE3 and OE6 are overexpression transgenic plants, and Ri6 and Ri9 are silencing transgenic plants;

FIG. 6 is a phenotype graph and a survival rate statistical graph of OsSKL2 transgenic rice under normal and salt treatment conditions; FIG. 6a shows the phenotype of wild type and transgenic plants under high salt treatment, and FIG. 6b shows the germination rate statistics. Respectively treating wild type and transgenic rice plants with the size of four weeks by 120mM and 140mM NaCl, wherein WT is a wild type plant, OE3 and OE6 are overexpression transgenic plants, and Ri6 and Ri9 are silent transgenic plants;

FIG. 7 is a phenotype graph and a survival rate statistical graph of OsSKL2 transgenic rice under normal and drought treatment; FIG. 7a is the phenotype of wild type and transgenic plants under drought treatment, and FIG. 7b is a statistical chart of germination rate. Wild type and transgenic rice plants with the size of four weeks are respectively treated by 20% PEG, WT is a wild type plant, OE3 and OE6 are overexpression transgenic plants, and Ri6 and Ri9 are silent transgenic plants.

Detailed Description

The present application will now be described in further detail with reference to the drawings, it should be noted that the following detailed description is given for illustrative purposes only and is not to be construed as limiting the scope of the present application, as those skilled in the art will be able to make numerous insubstantial modifications and adaptations to the present application based on the above disclosure.

1. Material

The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used therein are commercially available products unless otherwise specified.

2. Method of producing a composite material

2.1 acquisition of OsSKL2 Gene

Selecting a rice Nipponbare variety, extracting RNA and carrying out reverse transcription to obtain cDNA, designing primers SKL2-F-1 and SKL2-R-1 by taking the rice cDNA as a template and combining multiple cloning sites of a plant expression vector according to an OsSKL2 gene sequence and a rice genome database, and carrying out PCR amplification to obtain a PCR amplification product.

The primer sequence is as follows:

SKL2–F-1：ATGAGGATAGGGGCAGGGGCGAACA；

SKL2–R-1：TCAGATATTGGTCGGTGGGGCGTCG；

the PCR reaction program is: pre-denaturation at 98 deg.C for 10 min; denaturation at 98 ℃ for 20 s; annealing at 65 ℃ for 20 s; extending for 2min at 72 ℃ for 30 cycles; renaturation at 72 deg.C for 10 min; storing at 10 deg.C.

The PCR amplification product was detected by 2% by mass agarose gel electrophoresis, and the desired fragment was recovered and ligated to pEASY-T1 vector (purchased from all-grass gold Biotechnology Ltd., map of the vector is shown in FIG. 1) to obtain a ligated product. The ligation product was transformed into E.coli competent Trans5 α cells, and the plasmid was extracted. And (3) taking the extracted plasmid as a template, taking SKL2-F-1 and SKL2-R-1 as primers for PCR amplification verification, simultaneously carrying out detection by using Kpn I and Pst I double enzyme digestion plasmids, screening positive clones, sending the positive clones to China Dacron biology company for sequencing, comparing sequencing results by using Sequencher software, and obtaining results consistent with the prediction. The resulting recombinant plasmid was designated T-SKL 2.

2.2 construction of OsSKL2 Gene overexpression vector and silencing expression vector

pCAMBIA1301 (purchased from Shanghai Jielan Biotechnology Limited, and the vector map is shown in figure 2) is used as an original vector, a 35S promoter is connected between EcoRI and SacI enzyme cutting sites of a multiple cloning site of the pCAMBIA1301, and a section of NOS terminator is added between SphI and HindIII enzyme cutting sites of the pCAMBIA1301 to obtain a modified vector p 1. With Kpn I and PsT-SKL2 is subjected to double enzyme digestion by tI to obtain a target gene fragment, and simultaneously, p1 is subjected to double enzyme digestion by KpnI and PstI to obtain a vector fragment, and the two fragments are subjected to T₄And (3) ligase connection to construct a vector p1-OsSKL2 serving as an OsSKL2 gene over-expression vector of the transgenic rice. Rice cDNA is used as a template, an OsSKL2 partial gene fragment is amplified by using an OsSKL2 specific primer, the product is respectively subjected to double enzyme digestion by BamHI and HindIII, cloned into a pRNAi-Ubi vector subjected to the same enzyme digestion and converted into TOP10 competent cells, and an intermediate recombinant plasmid of a target fragment which is successfully assembled in a forward direction at one time is obtained through screening and identification by antibiotic (kanamycin) and PCR. The intermediate recombinant plasmid is amplified by using a carrier specific universal primer RNAi-Pst/RNAi-Mlu, a product is cloned into the intermediate recombinant plasmid with the same enzyme digestion after being subjected to double enzyme digestion by PstI and MluI, DH10B competent cells are transformed, and construction of a pRNAi-ubi SKL2 transformation vector is completed through kanamycin LB plate screening \ PCR \ enzyme digestion and sequencing identification. The vector is prepared by using BamH I to singly cut pRNAi-ubiSKL2, cloning into pRNAi-IP (pRNAi-IP vector after single cutting is treated by alkaline phosphatase) which is cut by the same enzyme, converting DH10B competent cells, screening by a kanamycin LB plate, carrying out enzyme cutting detection and sequencing identification, and completing the construction of the pRNAi-IP-SKL2 vector.

2.3 analysis of inducible expression Pattern of OsSKL2

Respectively treating wild rice middle flower 11 with the size of four sides with 100mM mannitol solution and 200mM NaCl solution, then selecting rice roots and leaves after 12h treatment, extracting RNA of the rice roots and leaves, respectively obtaining cDNA of the rice roots and leaves through reverse transcription, and determining the expression quantity of the treated roots and leaves through qRT-PCR.

The quantitative primers were designed as follows:

SKL2-F:TTGGCTTCGGAAGGAGTGGATT

SKL2-R:TTCCCGAGCTCACACTTCTACG

the qPCR reaction procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 20 s; annealing at 60 ℃ for 20 s; extension at 72 ℃ for 1min for 30 cycles. The results were subjected to mapping analysis.

The result is shown in figure 3, and the gene OsSKL2 is obviously induced and expressed by drought and high salt.

2.4 subcellular localization analysis of OsSKL2

First, a vector of pCAMBIA1305-SKL2 was constructed. The SKL2 and p1305 vectors were cleaved with SpeI and BamHI simultaneously, and the bands were recovered by gel electrophoresis, followed by recovery of pCAMBIA1305-SKL2 vector by overnight ligation with T4 ligase. Transferring the vector into a rice leaf protoplast by using a PEG transient transformation system, wherein the transformation system is as follows: 100 μ L protoplasts, 100 μ L pCAMBIA1305-SKL2 vector plasmid, and 200 μ L45% PEG. The transformation was induced for 30 minutes, diluted with 440. mu. L W5 solution and centrifuged, and 200. mu.L of WI solution was added and incubated in the dark for 12 to 18 hours, followed by observation by confocal laser microscopy.

The primers used in subcellular localization were as follows:

OsSKL2-F:ATGTTGGCCTCCACTTGCTTCTCCG

OsSKL2-R:TATGTTGGTGGGTGGTGCGTCGGA

as a result, as shown in FIG. 4, the gene OsSKL2 was found to be localized in chloroplasts.

2.5 Agrobacterium mediated acquisition and identification of OsSKL2 Gene overexpression and Silent expression plants

The methods for obtaining agrobacterium-mediated over-expressed plants and silenced expressed plants and the culture medium formulations used in the culture process are described in the literature: methods in Molecular Biology, vol.343: the method comprises the following steps of (1) Agrobacterium Protocols, 2/e and volume, and specifically comprises the following steps:

(1) obtaining of rice mature embryo induced callus

And selecting mature and full rice seeds, and shelling for later use. 1-2L of sterile water, 75% (mass fraction) alcohol, and sterile water prepared from hypochlorous acid and tween (the specific ratio: sterile water: hypochlorous acid 1: 1, total volume: tween 1mL:1ul) are prepared. Washing with 75% alcohol for about 5min, sterilizing with sterilized water for 15 min, separating for 7-8 min, defoaming with 75% alcohol for 3 times, washing with sterilized water for several times until the water becomes clear, placing the mature seed on sterilized filter paper, sucking water, culturing in callus induction culture medium at 26 + -1 deg.C; after 10-15 days, transferring the induced cream yellow callus to a subculture medium for subculture. Subculturing once every two weeks, selecting subculture for 5-7 days after subculturing twice, and using the callus with light yellow color for co-culture.

(2) Preparation of Agrobacterium culture solution for transformation of Rice

The p1-OsSKL2 and pRNAi-IP-SKL2 vectors are transformed into Agrobacterium LBA4404 to obtain recombinant Agrobacterium LBA4404/p1-OsSKL2 and LBA4404/pRNAi-IP-SKL 2. Recombinant Agrobacterium LBA4404/p1-OsSKL2 and LBA4404/pRNAi-IP-SKL2 were inoculated in YEP liquid medium (containing 50. mu.g/mL kanamycin and 50. mu.g/mL rifampicin), and shake-cultured at 28 ℃ to OD₆₀₀0.6-1.0; centrifuging at room temperature at 5000rpm for 5min to collect thallus, suspending in liquid co-culture medium, and adjusting thallus concentration to OD₆₀₀And (3) obtaining the agrobacterium suspension for co-culturing and transforming rice, wherein the suspension is 0.4.

(3) Rice nutritive organ infected by agrobacterium

Selecting tender and creamy callus, putting the callus into a 100mL sterile triangular flask together, and adding the prepared agrobacterium tumefaciens suspension; culturing at 28 deg.C and 220rpm on shaking table for 20-30 min; and (4) pouring out bacterial liquid, placing the infected callus on a culture dish containing sterile filter paper, absorbing excess bacterial liquid, transferring the infected callus onto a solid co-culture medium, and performing dark culture at 22 ℃ for 2-3 d.

(4) Selection culture

Cleaning the co-cultured callus with sterilized water until the water becomes clear, discarding the cleaning solution, transferring into a solution containing carbenicillin (100mg/ml), shaking and cleaning for more than 30min, drying with sterile filter paper, and drying in a culture dish containing sterile filter paper; then selecting the callus on a screening culture medium containing carbenicillin (500mg/ml) and hygromycin (50mg/L), and culturing the callus at 28 ℃ for about 30 days by illumination until new resistant callus is grown.

(5) Differentiation of resistant calli

Selecting new yellow callus from the screening culture medium, placing the new yellow callus on a differentiation culture medium containing 50mg/L hygromycin, performing dark culture at 28 ℃ for 3 days, transferring to the condition of 30 ℃ for full light culture for 15-30 days, and allowing green spots to appear; seedlings can be differentiated after 30-40 days.

(6) Rooting, strengthening seedlings and transplanting

Transferring the differentiated seedling to a rooting culture medium when the height of the seedling is 3cm, and culturing for 2-3 weeks; selectingHardening seedling with height of 15cm and developed root system at room temperature for 2-3 days, washing off culture medium with warm water, and culturing in water bucket containing rice culture soil. After the seedlings grow to be strong, the seedlings are moved into a paddy field to grow. 21 transgenic rice seedlings with 32 independent transformation events have 21 transgenic rice seedlings which simultaneously amplify target gene OsSKL2 fragments and hygromycin genes, and 8 transgenic rice seedlings with RNAi silence have 8 transgenic rice seedlings. T is₀After the generation transgenic plant is mature, harvesting T₁Seed generation, T₁Inbred propagation of seed generations to obtain T₂And (5) seed generation.

2.6 identification of transgenic Rice

In order to detect the transgenic rice, the total DNA of the extracted transgenic rice is used as a template, a target gene OsSKL2 fragment and a hygromycin gene are used as detection objects, primers are designed, PCR amplification is carried out, and the primers are used for preliminarily identifying transgenic plants.

The primers for detecting the OsSKL2 fragment of the target gene are as follows:

SKL2–F-2：ATGAGGATAGGGGCAGGGGCGAACA

SKL2–R-2：TCAGATATTGGTCGGTGGGGCGTCG

the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s; annealing at 60 ℃ for 30 s; extension at 72 ℃ for 1min for 35 cycles; total extension at 72 ℃ for 5 min.

Primers for detecting hygromycin gene:

Hyg-F：5’-TAGGAGGGCGTGGATATGGC-3’

Hyg-R：5’-TACACAGCCATCGGTCCAGA-3’

the PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 1min, and 34 cycles; total extension at 72 ℃ for 5 min.

Primers for detection of pRNAi-IP-SKL 2:

SKL2-Ri-N-F：TCCAGTCTGCACAAGTGAGC

SKL2-Ri-N-R：TCCAGCACAACACATTCAGC

pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s; annealing at 60 ℃ for 30 s; extension at 72 ℃ for 1min for 35 cycles; total extension at 72 ℃ for 5 min.

The results are shown in FIG. 5, and transgenic rice plants were obtained that were overexpressed (OE3 and OE6) and RNAi-silenced (Ri6 and Ri 9).

2.7 phenotypic identification and survival rate statistics of OsSKL2 transgenic rice under high-salt and drought treatment

The above identified rice plants with OsSKL2 transgene over-expression and silent expression were selected for study, and wild type middle flower 11 was used as a control for each group of treatments. Wild type and transgenic plants of four weeks size were treated with 120mM NaCl, 140mM NaCl and 20% PEG, respectively, and rehydrated for 1 day after drought treatment. And after treatment, photographing and survival rate statistics are respectively carried out.

The results are shown in fig. 6 and 7, after treatment with 120mM NaCl and 140mM NaCl, the survival rate of over-expressed plants was significantly higher, and the survival rate of silenced plants was significantly lower compared to wild type; similarly, the results of rehydration experiments after drought treatment also show that the survival rate of over-expressed plants is obviously higher, and the survival rate of silent plants is obviously lower; these results indicate that the OsSKL2 gene has salt resistance and drought resistance.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Sequence listing

<110> agriculture university of Anhui

<120> drought-resistant and salt-tolerant gene OsSKL2 of rice and application thereof

<141> 2021-12-16

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1128

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<213> Rice (Oryza sativa)

<400> 1

atgttggcct ccacttgctt ctccgccccg ccgccctcct cctcctcccc gtcaatcccg 60

acccacctcg ccactctctg ctgctgcttc aggcctcccg ctcgcccacc ttggcctcgc 120

tctcttcttc ttcttggtgc cttcccaccg ccgacccgcc ccctcccccg cgcctccgtc 180

tcgtcatcga ccgccccagc gaaagactac gagtttactg atggtggcgg agaggtggag 240

ctgaggctgg acataggaaa gctcggcatt gagaattcaa gagatgtatt tgttgacgtc 300

gatgatacgt ctctgttggt cagagccaag tcggatggga cactgcggac tttgatcaat 360

gttaaacaac tctttgatag gattaagtct tctgagacta tatggttcat tgatgaggat 420

caattggtag tgaatctaaa gaaagttgag caagagctga aatggcccga cattgatgaa 480

tcttgggaat cccttacttc tggaatcact cagcttttga cagggattag tgttcatatt 540

gttggtgatt ccacagatat aaacgaggca gttgctaaag aaatagctga gggaattggt 600

taccttccag tctgcacaag tgagctgcta gaaagtgcca ccgaaaagtc tattgacaaa 660

tggttggctt cggaaggagt ggattcggta gcagaagctg aatgtgttgt gctggaaagc 720

cttagcagcc atgttcgtac agtcgtagca actctggggg gaaagcaagg agcagctagc 780

agatttgata aatggcagta tcttcatgct ggatttacgg tttggttgtc ggtctccgat 840

gccagcgatg aagcttctgc caaagaagag gcccgtagaa gtgtgagctc gggaaatgtt 900

gcgtacgcga aagctgatgt agtagtgaag cttggtggat gggatccgga gtacacacga 960

gctgttgccc agggttgcct tgtggccttg aagcagctaa cattggcaga caagaagcta 1020

gcaggtaaga agagcctata catgaggctg ggatgccgag gggattggcc caacatcgag 1080

cctcccggct gggatcctga ctccgacgca ccacccacca acatatga 1128

<210> 2

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<213> Rice (Oryza sativa)

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Met Leu Ala Ser Thr Cys Pro Ser Ala Pro Pro Pro Ser Ser Ser Ser

1 5 10 15

Pro Ser Ile Pro Thr His Leu Ala Thr Leu Cys Cys Cys Pro Ala Pro

20 25 30

Pro Ala Ala Pro Pro Thr Pro Ala Ser Leu Leu Leu Leu Gly Ala Pro

35 40 45

Pro Pro Pro Thr Ala Pro Leu Pro Ala Ala Ser Val Ser Ser Ser Thr

50 55 60

Ala Pro Ala Leu Ala Thr Gly Pro Thr Ala Gly Gly Gly Gly Val Gly

65 70 75 80

Leu Ala Leu Ala Ile Gly Leu Leu Gly Ile Gly Ala Ser Ala Ala Val

85 90 95

Pro Val Ala Val Ala Ala Thr Ser Leu Leu Val Ala Ala Leu Ser Ala

100 105 110

Gly Thr Leu Ala Thr Leu Ile Ala Val Leu Gly Leu Pro Ala Ala Ile

115 120 125

Leu Ser Ser Gly Thr Ile Thr Pro Ile Ala Gly Ala Gly Leu Val Val

130 135 140

Ala Leu Leu Leu Val Gly Gly Gly Leu Leu Thr Pro Ala Ile Ala Gly

145 150 155 160

Ser Thr Gly Ser Leu Thr Ser Gly Ile Thr Gly Leu Leu Thr Gly Ile

165 170 175

Ser Val His Ile Val Gly Ala Ser Thr Ala Ile Ala Gly Ala Val Ala

180 185 190

Leu Gly Ile Ala Gly Gly Ile Gly Thr Leu Pro Val Cys Thr Ser Gly

195 200 205

Leu Leu Gly Ser Ala Thr Gly Leu Ser Ile Ala Leu Thr Leu Ala Ser

210 215 220

Gly Gly Val Ala Ser Val Ala Gly Ala Gly Cys Val Val Leu Gly Ser

225 230 235 240

Leu Ser Ser His Val Ala Thr Val Val Ala Thr Leu Gly Gly Leu Gly

245 250 255

Gly Ala Ala Ser Ala Pro Ala Leu Thr Gly Thr Leu His Ala Gly Pro

260 265 270

Thr Val Thr Leu Ser Val Ser Ala Ala Ser Ala Gly Ala Ser Ala Leu

275 280 285

Gly Gly Ala Ala Ala Ser Val Ser Ser Gly Ala Val Ala Thr Ala Leu

290 295 300

Ala Ala Val Val Val Leu Leu Gly Gly Thr Ala Pro Gly Thr Thr Ala

305 310 315 320

Ala Val Ala Gly Gly Cys Leu Val Ala Leu Leu Gly Leu Thr Leu Ala

325 330 335

Ala Leu Leu Leu Ala Gly Leu Leu Ser Leu Thr Met Ala Leu Gly Cys

340 345 350

Ala Gly Ala Thr Pro Ala Ile Gly Pro Pro Gly Thr Ala Pro Ala Ser

355 360 365

Ala Ala Pro Pro Thr Ala Ile

370 375

Claims

1. A drought-resistant and salt-tolerant gene OsSKL2 of rice is characterized in that the gene has a nucleotide sequence shown as SEQ ID NO. 1.

2. The application of the rice drought-resistant and salt-tolerant gene OsSKL2 in improving the drought resistance and salt tolerance of rice according to claim 1.

3. The use of claim 2, wherein the OsSKL2 gene is overexpressed, and has a positive regulation effect on drought resistance and salt tolerance of rice.

4. The coding protein of the rice drought-resistant and salt-tolerant gene OsSKL2 as claimed in claim 1, wherein the coding protein has an amino acid sequence shown as SEQ ID NO. 2.

5. A plasmid vector obtained by inserting the rice drought-resistant and salt-tolerant gene OsSKL2 of claim 1 into pEASY-T1 vector.

6. A genetically engineered host cell which is an E.coli competent Trans5 alpha cell comprising the plasmid vector of claim 5.

7. A method for obtaining a drought-resistant and salt-tolerant gene OsSKL2 of rice is characterized in that a nucleotide sequence of the gene OsSKL2 is used as a template to design a specific amplification primer, a Nipponbare variety of rice is used as a material, RNA is extracted and is reversely transcribed into cDNA, and the gene OsSKL2 is obtained through a PCR amplification technology.

8. The method for obtaining the rice drought and salt resistant gene OsSKL2 as claimed in claim 7, wherein the specific amplification primers for PCR amplification are:

SKL2–F-1：ATGAGGATAGGGGCAGGGGCGAACA；

SKL2–R-1：TCAGATATTGGTCGGTGGGGCGTCG。

9. an overexpression vector, which is a pCAMBIA1301 plant expression vector in which a 35S promoter, the gene OsSKL2 of claim 1 and an NOS terminator are sequentially linked to a multiple cloning site region.

10. A subcellular localization vector, characterized in that, the subcellular fusion vector is obtained by cutting the gene OsSKL2 and pCAMBIA1305 vector of claim 1 with SpeI and BamHI at the same time and then connecting, in particular pCAMBIA1305-SKL2, and the connection uses the upstream and downstream primers as follows:

OsSKL2-F：ATGTTGGCCTCCACTTGCTTCTCCG；

OsSKL2-R：TATGTTGGTGGGTGGTGCGTCGGA。