AU2020103419A4 - Application of AtSRT2 gene in improving salt tolerance of plants - Google Patents

Abstract

The invention discloses an AtSRT2 protein, which is characterized in that it is a protein with the amino acid sequence shown as SEQIDNO: 1 in the sequence table, or a protein derived from SEQIDNO: 2 by substituting, deleting or adding more than one amino acid residue in the amino acid sequence of SEQIDNO: 1, having the same activity as the amino acid sequence of SEQIDNO: 1. The encoding sequence of the AtSRT2 protein provided by the invention is introduced into a target plant by a genetic engineering method to obtain a transgenic plant with salt tolerance higher than that of the target plant, so that the salt tolerance of the plant is improved, which is mainly reflected as follows: under the salt treatment condition, compared with the wild type, the seed germination rate of the plant transferred with the encoding sequence of the AtSRT2 protein is obviously improved. The salt tolerance is enhanced, the plant grows better and is taller than the wild type, and the area of root and leaf is significantly higher than that of the wild type. The survival rate of plants is significantly improved. The invention plays an important role in breeding plants with salt tolerance and enhanced salt tolerance.

Description

Application of AtSRT2 gene in improving salt tolerance of plants

TECHNICAL FIELD

[01] The invention relates to the field of molecular genetics, in particular to an AtSRT2 protein and its coding sequence and application.

BACKGROUND

[02] Soil salinization has become a worldwide major environmental and resource problem that threatens human survival. According to the incomplete statistics of Food and Agriculture Organization of the United Nations (FAO), the salinized land area in the world is as large as 954 million hectares, which seriously restricts the sustainable development of agriculture and animal husbandry. The salinized land area in China is equivalent to 1/4 of the existing cultivated land. In recent years, with the continuous expansion of the salinized land area, the salinized soil area in China has reached 12 million hectares, making it a big country in the world (Lin Bing et al., 2005). Salt stress seriously inhibits the growth and development of plants, which is an important environmental stress factor causing crop yield reduction. Therefore, it is of great significance for global agricultural development and ecological environment improvement to improve the salt tolerance of crops by biotechnology, so as to expand the area of cultivated land and increase the yield of crops in saline-alkali land.

[03] In the process of plant evolution, in order to survive and multiply in the changing environment, plants have evolved corresponding molecular regulation networks to cope with salt stress. Under salt stress, many plant hormones such as abscisic acid, gibberellin, brassinolide and salicylic acid will participate in the regulation process. In addition, harmful ions are transported out of cells or isolated into vacuoles by activating SOS signaling pathway and vacuole membrane reverse transport channel NHX1, so as to control the accumulation of excessive ions in cytoplasm (Zhang and Blumwald, 2001;Yamaguchietal.,2013), as well as increase the synthesis of osmotic protective agents such as proline, polyhydric alcohols, carbohydrate and methylamine, and regulate the growth of plants under salt stress through osmotic adjustment. Above methods are all important ways for plants to cope with salt stress (Deinleinetal.,2014).

[04] In recent years, with the development of epigenetics, it has been found that chromosome histone modification plays a role in the adaptability of plants to various stresses. Studies have shown that when salt stress occurs, in contemporary plants, chromosome modification is involved in salt-resistant response.

[05] Arabidopsis thaliana (L.) Heynh., cultured under normal conditions after acclimating with slight salt stress in seedling stage, has no salt stress growth stage in later stage, and there is no visible phenotypic difference between the acclimated plants and the control. After salt stress treatment, it is found that the induced expression of HKT1 in the acclimated plants is higher than that in control group, and the accumulation of Na' is lower, showing more salt resistance. The analysis of four chromosome modifications in the whole genome by ChIP-seq shows that the modification distribution of H3K27me3 changed after salt stress acclimation (Sanietal.,2014). Song et al. (2012) reports that salt stress can affect DNA methylation status of promoters and coding regions of four transcription factors in soybean, indicating that epigenetic modification of these genes can enhance salt tolerance of plants. The research of Zhao Liang et al. (2011) shows that histone arginine methyltransferase SKB1 can affect the expression of key genes of ABA signaling pathway by changing the level of histone H4R3sme2, thus mediating the salt tolerance of plants.

[06] In the epigenetic regulation of plant salt tolerance, DNA methylation and histone methylation are mainly studied, while other modifications such as histone acetylation are seldom studied. AtSRT2 is a histone deacetylase, while at present, there is no report on the role of AtSRT2 in mediating plant salt tolerance. In this study, it has found that AtSRT2 could mediate the regulation of plant salt tolerance, and overexpression of AtSRT2 gene could improve the salt tolerance of Arabidopsis thaliana, especially of seed germination. Seed germination is a key period in the process of plant growth and development, which directly affects the whole subsequent development stage of plants. In this period, it is very sensitive to the environment and more vulnerable to salt stress. The invention has important significance for molecular improvement of salt tolerance of crops.

SUMMARY

[07] Technical requirements

[08] A technical problem to be solved by the invention is to provide an AtSRT2 protein.

[09] The technical problem to be solved by the present invention is to provide the coding sequence of above-mentioned protein fragment.

[010] The technical problem to be solved by the present invention is to provide the application of the gene fragment in improving the salt tolerance of plants.

[011] The invention is realized as follows.

[012] An AtSRT2 protein is a protein with the amino acid sequence shown as SEQIDNO: 1 in the sequence table, or a protein derived from SEQIDNO: 2 by substituting, deleting or adding more than one amino acid residue in the amino acid sequence of SEQIDNO: 1, having the same activity as the amino acid sequence of SEQIDNO: 1.

[013] The coding sequence of AtSRT2 protein is one of the following nucleotides:

[014] (1) a nucleotide with the nucleotide sequence shown as SEQIDNO: 2 in the sequence table;

[015] (2) a polynucleotide with the amino acid sequence shown as SEQIDNO: 1 in the coding sequence table;

[016] (3) a nucleotide with the nucleotide sequence which can hybridize with the nucleotide sequence defined by SEQIDNO: 2 in the sequence table under high stringency conditions.

[017] The application of coding sequence of AtSRT2 protein in improving salt tolerance of plants.

[018] The AtSRT2 protein of the invention can be synthesized artificially, or its coding gene can be synthesized first and then expressed biologically.

[019] The encoding sequence of the AtSRT2 protein of the present invention can be obtained by deleting codons of one or several amino acid residues in the DNA sequence shown in SEQIDNO: 2 in the sequence table, and/or carrying out missense mutation of one or several base pairs, and/or connecting the encoding sequence of the tag shown in SEQIDNO: 1 at its 5' end and/or 3' end.

[020] SEQIDNO: 2 consists of 1131 nucleotides. The whole SEQIDNO: 2 is the coding sequence of AtSRT2 protein, and SEQIDNO: 1 consists of 376 amino acids.

[021] The high stringency condition is hybridization and washing membrane in the solution of 0.1xSSPE (or 0.1xSSC) and 0.1 %SDS at 65C.

[022] Recombinant expression vectors, expression cassettes, transgenic cell strains or recombinant bacteria containing the gene should belong to the protection scope of the present invention.

[023] Existing expression vectors can be used to construct recombinant expression vectors containing the genes. The expression vectors include binary Agrobacterium vectors and vectors which can be used for micro-bomb bombardment. The expression vector can also contain the 3' untranslated region of foreign gene, that is, it contains polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can guide polyadenylation to be added to the 3' end of the mRNA precursor. When using the gene to construct a recombinant expression vector, any enhanced promoter or constitutive promoter can be added before the transcription of initiation nucleotide, besides, they can be used alone or in combination with other promoters. In addition, when using the gene of the present invention to construct a recombinant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used. These enhancers can be ATG initiation codons or adjacent region start codons but must be the same as the reading frame of the coding sequence to ensure the correct translation of the whole sequence. The sources of the translation control signal and the initiation codon are extensive, which can be natural or synthetic. Translation initiation region can be derived from transcription initiation region or structural gene. In order to facilitate identification and screening, the expression vectors used can be processed, such as adding genes encoding enzymes or luminescent compounds that can produce color changes, antibiotic markers with resistance or anti-chemical agent marker genes, etc. It can also be screened directly according to phenotype without adding any selective marker genes.

[024] The recombinant expression vector can be a recombinant plasmid obtained by inserting the gene into the multiple cloning site of pBI121 plasmid.

[025] Beneficial effects

[026] The encoding sequence of the AtSRT2 protein provided by the invention is introduced into a target plant by a genetic engineering method to obtain a transgenic plant with salt tolerance higher than that of the target plant, so that the salt tolerance of the plant is improved, which is mainly reflected as follows: under the salt treatment condition, compared with the wild type, the seed germination rate of the plant transferred with the encoding sequence of the AtSRT2 protein is obviously improved; the salt tolerance is enhanced, the plant grows better and is taller than the wild type, and the area of root and leaf is significantly higher than that of the wild type; the survival rate of plants is significantly improved. The method plays an important role in breeding plants with salt tolerance and enhanced salt tolerance.

BRIEF DESCRIPTION OF THE FIGURES

[027] Figure. 1 shows that the expression of AtSRT2 gene during seed germination is induced by salt stress.

[028] Figure. 2 shows that AtSRT2 subcellular localization signal is affected by salt stress.

[029] Figure. 3 is a schematic structural diagram of the recombinant plasmid PBI121-AtSRT2.

[030] Figure. 4 is the screening of AtSRT2 transgenic positive plants.

[031] (WT represents wild Arabidopsis thaliana, Col/pBI121 is Arabidopsis thaliana transformed into pBI121 empty vector, OE-1, OE-2 and OE-3 respectively represent three AtSRT2 transgenic strains)

[032] Figure. 5 is a comparison of the seed germination rates of three AtSRT2 transgenic strains and wild Arabidopsis thaliana under salt treatment (WT represents wild Arabidopsisthaliana, OE-1, OE-2 and OE-3 respectively represent three AtSRT2 transgenic strains)

[033] Figure. 6 is a graph showing the plant growth phenotype of three AtSRT2 transgenic strains and wild Arabidopsis thaliana under salt treatment (WT represents wild Arabidopsisthaliana, OE-1, OE-2 and OE-3 respectively represent three AtSRT2 transgenic strains)

[034] Figure. 7 is a comparison of plant morphology and survival rate between three AtSRT2 transgenic strains and wild Arabidopsis thalianaunder salt treatment.

[035] (WT represents wild Arabidopsis thaliana, OE-i, OE-2 and OE-3 respectively represent three AtSRT2 transgenic strains)

DESCRIPTION OF THE INVENTION

[036] The present invention can be better understood from the following embodiments. However, those skilled in the art will easily understand that the embodiments described are only for illustrating the invention and should not and will not limit the invention described in detail in the claims.

[037] Materials and reagents used in the following embodiments can be obtained from comMercial sources unless otherwise specified.

[038] Arabidopsis thaliana L. ecotype Columbia0: can be ordered from Arabidopsis database, http://www.arabidopsis.org/.

[039] Arabidopsis thaliana is planted in the phytotron of Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. The temperature of the phytotron is set at 21°C, and the long sunshine is 16h light/8h dark, and the short sunshine is 10h light/14h dark.

[040] pBIl21 vector is purchased from Clontech company, with catalog number of 6081-1.

[041] Embodiment 1. The expression of AtSRT2 induced by salt stress during seed germination

[042] 1. Extraction of total RNA from Arabidopsis thaliana

[043] The wild Arabidopsis thaliana ColumbiaO seeds are treated with 70% (volume fraction) alcohol for 3min in clean bench, then washed with sterile water twice for 1min each time, sterilized with 5% (mass/volume ratio) sodium hypochlorite for min, and then washed with sterile water twice for one minute each time. In the control group, sterile seeds are sown in MSO liquid medium with a pipette, and then transferred to tissue culture room after 3 days of vernalization at 4C. Samples are taken at Oh, 6h, 12h, 24h, 36h and 48h respectively. At the same time, sterile seeds in salt treatment group are sown in MSO liquid medium containing 60mM NaCl with a pipette, and then transferred to tissue culture room after 3 days of vernalization at 4C. Samples are taken at Oh, 6h, 12h, 24h, 36h, 48h, 60h and 72h.

[044] Total RNA is extracted from the treated wild Arabidopsis thaliana by TRIzol method. After the extracted RNA is qualified by 2% agarose gel electrophoresis, the total RNA is reverse transcribed into cDNA by reverse transcriptase (TaKaRa company) and stored at -20°C for later use.

[045] 2. Detection of the AtSRT2 gene expression by RT-PCR

[046] Specific primers AtSRT2-F1 and AtSRT2-R1 are designed according to AtSRT2 gene (AT5G09230, Arabidopsis thaliana genome locus number; http://www.arabidopsis.org/)sequence, and the target fragment is 273bp. With Arabidopsis thalianagene as internal reference, primers AtActin-F and AtActin-R are designed with the target fragment of 227bp.

[047] The cDNA processed in different time periods obtained in step 1 are diluted to appropriate concentration and used as template for real-time quantitative PCR determination, which is performed according to instructions of Real Master Mix (SYBR Green)PCR kit of TIANGEN company. The reaction system includes 4pl of 2.5xReal Master Mix, 0.5 pl of each upstream and downstream primers, 4.5 p 1 of ddH204, and Ip of template. The procedure of PCR amplification is as follows: pre-denaturation at °C for 3min, denaturation at 95C for 20s, annealing at 53C for 20s, extension at 72°C for 20s, 40 cycles, fluorescence collection in the third step of each cycle, and 3 replicates for each sample.

[048] AtSRT2-F: 5'-TTCCGTCGCGAAGTATGTACC-3' (At5GO9230: 53 73bp, the 53rd-73rd bits of SEQIDNO: 2 in the sequence table)

[049] AtSRT2-R: 5'-CTGTGCTAACCCCAGCTCCAGTC-3' (At5G09230: 303-325bp, the reverse complementary sequence of the 303rd-325th bits of SEQIDNO: 2 in the sequence table)

[050] AtActin-F: 5'-CCCCTGCTATGTATGTGGCTAT-3'

[051] AtActin-R: 5'-TGCTGTGGTGGTGAAAGAGTAA-3'

[052] The results of RT-PCR analysis show that the dissolution curves are single peaks,the amplification products have good specificity, and the fluorescence curves could reflect the amplification results well. The expression of AtSRT2 gene in different periods during seed germination under salt treatment is statistically analysed. The results are shown in Fig.1, indicating that it takes 48 hours for seeds to germinate normally under non-treatment conditions, and the expression of AtSRT2 is stable during the whole process without obvious up-down change. Under the condition of mM NaCl treatment, seed germination is delayed, which takes 72 hours. The expression of AtSRT2 increases gradually with the extension of treatment time, reaching the highest value at 48 hours, which is 9.21 times of the initial transcription level, and then decreases gradually. The above results indicate that the expression of AtSRT2 is induced by salt stress during seed germination.

[053] Embodiment 2 Subcellular localization of protoplasts

[054] 1. Construction of subcellular localization vector

[055] According to CDS sequence of AtSRT2 gene provided on TAIR website, specific primers AtSRT2-F2 and AtSRT2-R2 are designed to amplify the full length of AtSRT2 gene (stop codon removed). The Infusion enzyme is used to link the target fragment to the subcellular localization vector 16318GFP digested by BamHI enzyme. The reaction system is as follows: 2 1 of Iinfusion HD Enzyme Promix, 5 1 of 16318 GFP digestion product, 3 l of PCR amplification product, and reaction at 50°C for 15 minutes. Transformation of Escherichia coli Top10 is performed by heat shock after ligation reaction. The recombinant plasmid is detected by PCR. The positive clones are sequenced. The recombinant plasmid 116318-AtSRT2-GFP is obtained by extracting the plasmid.

[056] Primer sequence is:

[057] 16318-AtSRT2-GFP-F:

[058] 5'-TATCTCTAGAGGATCCATGCTTTCTATGAACATGAGAAG-3' (the underlined part is the recognition site of BamHI, and the subsequent sequence is the 1st-23rd of SEQIDNO: 2)

[059] 16318-AtSRT2-GFP-R:

[060] 5'-TGCTCACCATGGATCCGAGAGCTGGGACACTGAGCG-3' (the underlined part is the recognition site of BamHI, and the subsequent sequence is the reverse complementary sequence of 1109th-I128th of SEQIDNO: 2)

[061] 2. Preparation of protoplast

[062] 1) Arabidopsis thaliana grown for about 2-3 weeks is taken as the test material(Arabidopsisthalianais small, and the prepared protoplast has good integrity).

[063] 2) Under the condition of low light, taking the leaves and cut them into strips of 0.5-1mM (do not cut them completely, but keep the strips connected at the same leaf edge, so that the protoplast can be cracked more fully).

[064] 3) Adding 10-20ml enzymolysis solution, covering it with tin-platinum paper, and keeping it away from light at room temperature at 45 rpm for 3 hours.

[065] 4) Carefully filtering with 100-150 mesh steel filter screen.

[066] 5) Taking 1OOg to centrifuge at room temperature for 1 minute (Brake, the speed is adjusted to 2 to prevent the speed from rising and falling rapidly) and removing the supernatant (keep a small amount of liquid).

[067] 6) Gently mixing first, then adding 1ml W5 to resuspend. Gently mixing again, then performing ice bath for 30 minutes, centrifuging 1OOg for 1 minute (same as above) and removing supernatant (same as above).

[068] 7) Mixing gently first, then adding a proper amount of MMG to resuspend.

[069] 3. Conversion at room temperature

[070] 1) Adding 10d of 16318- AtSRT2-GFP (10-20tg plasmid DNA), generally

[tg.

[071] 2) Adding 100d1of protoplast and mixing well.

[072] 3) Adding 1I0d1PEG/Ca solution (PEG should keep a constant volume) and mixing well.

[073] 4) Incubating at 23C for 30 minutes.

[074] 5) Adding 440d1W5 solution and mixing it gently.

[075] 6) Centrifuging 100 g for 1 minute (same as above) and removing PEG from supernatant.

[076] 7) Adding 100d1of W5 to resuspend, and then adding 0.9ml W5 (salt treatment experiment, adding NaCl to W5 until the final concentration is 60mM).

[077] 8) Culturing in dark at 23C overnight.

[078] 9) Observe GFP signal under confocal microscope.

[079] As shown in Fig. 2, AtSRT2 is located in the cell membrane and nucleus under non-treatment conditions (Fig. 2-A). Under the condition of 60mM NaCl treatment, the positioning signal of AtSRT2 membrane disappears, only can get nuclear positioning signal (Fig. 2-B). The nuclei are stained with DAPI, and it is found that the GFP signal of AtSRT2 is completely overlapped with the stained part of DAPI, which indicates that AtSRT2 is localized in the nuclei under the condition of 60mM NaCl treatment. The above results indicate that the subcellular localization of AtSRT2 changes with NaCl treatment, and AtSRT2 responds to NaCl treatment.

[080] Embodiment 3 Cloning of AtSRT2 gene and obtaining over-expression strain

[081] 1. Cloning of AtSRT2 gene and construction of recombinant expression vector pB121-AtSRT2

[082] The mature wild Arabidopsis thaliana ColumbiaO is used as experimental material, and the total RNA is extracted by TRIzol method. The qualified RNA is reverse transcribed into cDNA by reverse transcription kit of TaKaRa Company and stored at -20°C for later use. According to CDS sequence of AtSRT2 gene provided on

TAIR website, specific primers AtSRT2-F2 and AtSRT2-R2 are designed to amplify the full length of AtSRT2 gene. The reaction system includes 25 1 of 2x GC Buffer, 4 1 of dNTPMix, each 1 1 of upstream and downstream primers, 0.3 1 of Primerstar, 16.7 1 of ddH20 and 2 1 of template cDNA (all the above PCR reagents come from PCR amplification kit of TaKaRa company). The PCR amplification procedure is pre denatured at 94C for 10min, denatured at 94C for 30s, annealed at 49C for 45s,

extended at 72C for 1min, 40 cycles, finally annealed at 72C for10min and stored at

4°C.

[083] pBI121-AtSRT2-F: 5' CCCCCGGGATGCTTTCTATGAACATGAGAAG-3' (the underlined part is the recognition site of SmaI, and the subsequent sequence is the 1st-23rd of SEQIDN: 2)

[084] pBIl21-AtSRT2-R: 5' GGACTAGTCTAGAGAGCTGGGACACTGAGCG-3' (the underlined part is the recognition site of Spel, and the subsequent sequence is the reverse complementary sequence from 1109th to 1131st of SEQIDNO: 2)

[085] PCR products are subjected to agarose gel electrophoresis, and the target DNA is recovered and purified by DNA gel recovery kit of TaKaRa company. The PCR products are inserted into the cloning vector using PEasy-Bluntcloning Vector system of TIANGEN Company. The reaction system is 10 : 8[ 1PCR product, 21 vector. After standing at room temperature for 15min, adding the ligation product to 100[1 TOP10 competent cells, mixing them well in ice bath for 30s, and putting it on ice for 2min immediately after heat shock at 42C for 90s. Then, 500[ 1of LB culture solution

balanced to room temperature is added and cultured in a shaker at 37C for 1 hour at

200rpm. After smearing the turbid bacterial liquid on LB medium containing Amp resistance with glass rod, it is cultured overnight in a constant temperature incubator at 37°C. White monoclonal antibody is selected, and the bacterial liquid is detected by

PCR using primers AtSRT2-F2 and AtSRT2-R2 (the target fragment size is about 1131bp). The recombinant plasmid is obtained by shaking the plasmid of the strain identified correctly by PCR, using restriction endonucleases SmaI and Spel to cut the plasmid, and connecting the target band of about 1131bp with the large fragment of pBI121 vector which had undergone the same double digestion. Sequencing confirms that the recombinant plasmid inserted into the DNA fragment (AtSRT2 gene) shown in SEQIDNO: 2 in the sequence table at the digestion sites SmaI and Spel of pBIl21 is pBIl21-AtSRT2 (Fig. 3). In the recombinant expression vector pBIl21-AtSRT2, the promoter that starts the expression of AtSRT2 gene is 35s promoter. SEQIDNO: 2 is the coding sequence of the protein (AtSRT2 protein) shown in SEQIDNO:1.

[086] 2. Acquisition and identification of Arabidopsis thaliana with transgenic AtSRT2

[087] The recombinant expression vector pBIl21-AtSRT2 (or pBIl21 vector) obtained in step lis transformed into Agrobacterium C58C1 (purchased from Biovector CO.,LTD, with item number Biovector009) by freeze-thaw method (the method of transforming Agrobacterium by freeze-thaw method, referring to Holsters M, deWaele D, Depicker A. Transfection and transformation of Agrobacterium tumefaciens. MolGenGenet,1978,183:181-187).PCR identification is carried out with primers AtSRT2-F2 and AtSRT2-R2, and the recombinant Agrobacterium containing the DNA fragment (about 1131bp) shown in SEQIDNO: 2 in the sequence table is named C58C1/pBI121-AtSRT2. The recombinant Agrobacterium transformed into pBI121 empty vector is named C58C1/pBIl21. The method of Agrobacterium inflorescence infection (Bechtold N et al., (1993) In plant an Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants.C.R.Acad.Sci.316:1194-1199) is used to transform above recombinant Agrobacterium into Arabidopsisthalianaecotype Columbia0. After screening with MSO solid medium containing 50mg/mL Kan, 9 TO plants with AtSRT2 gene are screened out, of which three are named OE-1, OE-2 and OE-3 (three TO plants with AtSRT2 gene are shown in Fig. 4-A). Transplanting the screened positive plants to nutrient soil for cultivation in greenhouse, screening each generation of seeds on MSO solid medium containing 50mg/mL Kan, and performing functional identification when propagating to T3 generation. Furthermore, the transgenic Arabidopsisthalianaseedlings of T3 generation are detected by PCR, using the genomic DNA of the transgenic Arabidopsisthalianaseedlings as template, and the primers for PCR detection are AtSRT2-F2 and AtSRT2-R2. PCR detection results show that the fragments with the length of about 113lbp are amplified from the three different strains with AtSRT2 gene. However, the same primers are used for PCR detection of wild-type Arabidopsis Columbia0 plants and plants transferred into pBIl21 empty vector, and the amplified fragments are not obtained (Fig. 4-B).

[088] Embodiment 4 Identification of salt tolerance of Arabidopsis thalianawith transgenic AtSRT2

[089] 1. Detection of salt tolerance during germination

[090] The following three Arabidopsis thaliana plants are used as experimental materials: wild Arabidopsis thalianaColumbia0 (WT), three T3-generation transgenic AtSRT2 strains (OE-1, OE-2 and OE-3), plants transferred into pBIl21 empty vector. Seeds are seeded on MSO solid medium and MSO solid medium containing 120mM NaCl and 150mM NaCl after sterile treatment, and then are put into tissue culture room for growth after 3 days vernalization at 4C. After 2 days of treatment, the germination rate of seeds is counted. Under the non-treated and treated conditions, there is no significant difference between the strains transferred into the empty vector pBI121 and the wild type. However, as shown in Fig. 5, the germination rate of wild-type seeds is slightly lower than that of AtSRT2 overexpressed strains under non-treatment conditions, and the germination rates of wild-type seeds and AtSRT2 over-expressed strains tend to be consistent after 4 days of light culture, both reaching more than 96%. Under NaCl treatment, both wild mutants and AtSRT2 overexpressed strains showed delayed seed germination and decreased seed germination rate. Under the treatment of 120mM and 150mM NaCl, the seed germination rate of the over-expressed strain of AtSRT2 is delayed by about 1 day compared with that under normal growth conditions, but the final seed germination rate is equivalent to that of the non-treatment. Besides, the germination rate of wild type seeds decreases by 39.37% and 58.1% under 120mM and 150mM NaCl treatment compared with that of non-treatment. The above results indicate that the transgenic strain with AtSRT2 gene significantly improves the seed germination rate and final seed germination rate under salt treatment and improves the salt tolerance of seeds during germination.

[091] 2. Detection of salt tolerance at seedling stage

[092] The following three Arabidopsis thaliana plants are used as experimental materials: wild Arabidopsis thalianaColumbiaO (WT), three T3-generation transgenic AtSRT2 strains (OE-1, OE-2 and OE-3), plants transferred into pBI121 empty vector. Seeds are germinated and cultured in MSO solid medium for 5 days, then transferred to MSO solid medium containing 90mM NaCl. After treatment for 10 days, phenotypes of all materials are observed, and root and leaf area scanning, and data statistical analysis are carried out. Under the non-treated and treated conditions, there is no significant difference between the strains transferred into the empty vector pBI121 and the wild type. However, as shown in Fig. 6, under non-treatment conditions, the size of the transgenic strain with AtSRT2 gene is equivalent to that of the wild type, and the results of root and leaf area scanning show that there is no significant difference among materials. Under the treatment of 90mM NaCl, the growth and development of all experimental materials are seriously affected, and the plants become short. Relatively speaking, the wild type is more seriously affected than the transgenic strain with AtSRT2 gene. The results of root and leaf area scanning shows that the leaf area of transgenic strains with AtSRT2 gene is significantly higher than that of wild type, and the leaf area of transgenic strains OE-1 and OE-3 with AtSRT2 gene is significantly higher than that of wild type.

[093] At the same time, the experimental materials are germinated and cultured in MSO solid medium for 8 days, and then transferred to soil for cultivation in greenhouse. After 10 days of cultivation, they are treated with 150mM NaCl, and after 21 days of treatment, the survival rate of plants is counted. As shown in Fig. 7, before 150mM NaCl treatment, there is no obvious difference in phenotypes of all experimental materials. After 150mM NaCl treatment, leaves of all experimental materials begin to turn yellow, and some plants dies. Relatively speaking, the wild type is more seriously affected by salt stress than the transgenic AtSRT2 strains. The statistical results of plant relative survival rate show that under 150 mm NaCl treatment, the relative survival rates of OE-1, OE-2 and OE-3 are 78.1%, 81.8% and 68%, respectively, which is significantly higher than that of the wild type (26.8%).

[094] The results of the above embodiments show that compared with the wild type, the transgenic strain with AtSRT2 gene significantly improves the ability of plants to maintain normal growth and development under salt stress and improves the relative survival rate of plants.

[095] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, in keeping with the broad principles and the spirit of the invention described herein.

[096] The present invention and the described embodiments specifically include the best method known to the applicant of performing the invention. The present invention and the described preferred embodiments specifically include at least one feature that is industrially applicable

Claims (3)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An AtSRT2 protein, characterized in that it is a protein with the amino acid sequence shown as SEQIDNO: 1 in the sequence table, or a protein derived from SEQIDNO: 2 by substituting, deleting or adding more than one amino acid residue in the amino acid sequence of SEQIDNO: 1, having the same activity as the amino acid sequence of SEQIDNO: 1.

2. The coding sequence of AtSRT2 protein according to claim 1, characterized in that it is one of the following nucleotides:

(1) a nucleotide with the nucleotide sequence shown as SEQIDNO: 2 in the sequence table;

(2) a polynucleotide with the amino acid sequence shown as SEQIDNO: 1 in the coding sequence table;

(3) a nucleotide with the nucleotide sequence which can hybridize with the nucleotide sequence defined by SEQIDNO: 2 in the sequence table under high stringency conditions.

3. An application of the coding sequence of AtSRT2 protein according to claim 2 in improving salt tolerance of plants.