CN115772212A - Alfalfa chloroplast MsSAP22 gene and application thereof in improving drought resistance of plants - Google Patents

Abstract

The invention discloses an alfalfa chloroplast MsSAP22 gene and application thereof in improving drought resistance of plants, wherein a nucleotide sequence of the MsSAP22 gene is shown as SEQ ID No.1, and an amino acid sequence of a coding protein thereof is shown as SEQ ID No. 2. The MsSAP22 expression protein is positioned in chloroplast. The overexpression of the MsSAP22 gene can enhance the drought tolerance of arabidopsis thaliana, is favorable for explaining the effect of the MsSAP22 gene on the osmotic stress resistance response from a molecular mechanism, and has important significance on the directed genetic improvement of alfalfa.

Description

Alfalfa chloroplast MsSAP22 gene and application thereof in improving drought resistance of plants

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an alfalfa chloroplast MsSAP22 gene and application thereof in improving drought resistance of plants.

Background

Alfalfa (Medicago sativa subsp. Sativa) plays a great role as the most important leguminous pasture in the development of grass husbandry and ecological economic construction in China (Yang Qingchuan, etc., 2016). Arid and semiarid regions in northwest and north China are main alfalfa production regions in China, and the arid regions are dry, rain-less and water-deficient and become key bottlenecks affecting alfalfa yield and geographical distribution (Chen Jingdeng, 2020). In addition, the shortage of drought-resistant alfalfa varieties and the rapid increase of the demand on high-quality pasture in China cause the long-term dependence on import of alfalfa hay and seeds, and the problem of influencing the development of the grass husbandry in China is caused. Therefore, the excavation of the drought-resistant functional gene of the alfalfa and the analysis of the physiological and molecular mechanism of the response drought of the gene have important theoretical significance and practical value for promoting the genetic improvement of the high drought-resistant alfalfa and the development of the grass and animal husbandry in China.

In the practical research, because alfalfa has the complex characteristics of polyploidy inheritance, inbreeding depression, cross pollination, self incompatibility and the like, the analysis of the genome and the cultivation of a new variety of alfalfa are greatly hindered. Alfalfa is the main pasture grass in arid and semi-arid regions in northwest and north China, and although the alfalfa has certain drought resistance, the alfalfa has weak drought resistance. With the increasing scarcity of water resources, drought stress has become a major factor limiting the yield and quality of alfalfa. In recent years, researchers have studied alfalfa in response to drought stress in various fields such as morphology, physiology, proteomics, and transcriptomics (Ma et al, 2017, luo et al, 2019, wang et al, 2020), but still has a serious lag compared to other model plants and important crops. Therefore, the basic research of alfalfa responding to drought stress is urgently needed to be enhanced, and a theoretical basis is laid for improving the drought tolerance of alfalfa and solving the problem that high-quality alfalfa in China seriously depends on import.

Disclosure of Invention

The invention aims to provide an alfalfa chloroplast MsSAP22 gene and application thereof in improving drought resistance of plants.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the alfalfa chloroplast MsSAP22 gene has a nucleotide sequence shown in SEQ ID No.1, and the nucleotide sequence consists of 366 bases.

An amino acid sequence of the alfalfa chloroplast MsSAP22 protein is shown in SEQ ID No. 2. The sequence consists of 121 amino acid residues.

The overexpression vector containing the alfalfa chloroplast MsSAP22 gene also falls into the protection scope of the invention, and the overexpression vector selected by the invention is an agrobacterium overexpression vector.

The most important invention point of the invention is that the gene can improve the drought tolerance of plants through osmotic stress resistance experiments. That is, the gene or protein or overexpression vector plays an important role in improving drought tolerance of plants.

The plants include alfalfa, tobacco and arabidopsis thaliana, but are not limited to alfalfa, tobacco and arabidopsis thaliana, and the same technical effect can be obtained when the overexpression vector of the MsSAP22 gene can be transferred into the plants by utilizing a transgenic technology.

The drought tolerance of the invention comprises: transgenic plants are more tolerant than wild type plants under osmotic stress than wild type plants. The concrete expression is as follows: on 1/2 MS medium containing 300mM mannitol and 30. Mu.M ABA, both wild type and transgenic lines were inhibited in root growth, but the transgenic lines had significantly higher root length and lateral root number than the wild type. The drought resistance of transgenic Arabidopsis thaliana in soil was also evaluated. Firstly, col-0 and a transgenic strain are germinated on a normal 1/2 MS culture medium, and then the germinated arabidopsis seedlings are transplanted into soil, wherein the soil comprises turfy soil and vermiculite (3: 1). After the seedlings grow for about 20 days, watering the flowerpot until the seedlings are saturated, performing drought treatment, setting 3 biological repetitions for each treatment, observing the phenotype, and performing drought treatment on wild type and transgenic lines for 12 days, wherein the transgenic arabidopsis begins to have wilting and dry-out phenomena, but the wild type arabidopsis is completely dry-out. At the moment, all arabidopsis thaliana are subjected to rehydration treatment, and the result shows that the survival rate of the transgenic arabidopsis thaliana after rehydration is significantly larger than that of a wild type, so that the MsSAP22 gene enhances the drought resistance of the transgenic arabidopsis thaliana.

The invention also provides a plant breeding method, which is characterized in that the method is (1) or (2):

(1) Obtaining a plant with drought tolerance stronger than that of a target plant by increasing the activity of MsSAP22 protein in the target plant;

(2) By promoting the expression of the MsSAP22 gene in the target plant, the plant with stronger drought tolerance than the target plant is obtained.

Wherein, the target plant is Arabidopsis thaliana.

A target gene (also called a target gene) is used for gene recombination, modification of a recipient cell trait, and acquisition of a gene of a desired expression product in genetic engineering design and operation. Either the organism itself or from a different organism.

In the present invention, there is no particular limitation on the plant or the target plant suitable for use in the present invention, as long as it is suitable for carrying out a gene transformation operation, such as various crops, flowering plants, or forestry plants. The plant may be, for example (without limitation): dicotyledonous, monocotyledonous, or gymnosperm.

As a preferred mode, the "plant" includes but is not limited to: alfalfa, tobacco, arabidopsis, all genes with or homologous to the gene are suitable.

As used herein, "plant" includes whole plants, parent and progeny plants thereof, and various parts of the plant, including seeds, fruits, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, having the gene or nucleic acid of interest in each of these various parts. Reference herein to "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the foregoing comprises a gene/nucleic acid of interest.

The present invention includes any plant cell, or any plant obtained or obtainable by the methods therein, as well as all plant parts and propagules thereof. The present patent also encompasses transfected cells, tissues, organs or whole plants obtained by any of the foregoing methods. The only requirement is that the progeny exhibit the same genotypic or phenotypic characteristics and that the progeny obtained using the methods of this patent have the same characteristics.

The invention also extends to harvestable parts of a plant as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. It also relates to other post-harvest derivatives of the plant, such as dry granules or powders, oils, fats and fatty acids, starches or proteins. The invention also relates to food or food additives obtained from the relevant plants.

The invention has the advantages that:

the invention discloses an alfalfa MsSAP22 gene for the first time, and determines the amino acid sequence of the gene. And the MsSAP22 expression protein was found to be localized in chloroplasts.

The overexpression of MsSAP22 can enhance the drought tolerance of Arabidopsis, is favorable for explaining the effect of the MsSAP22 gene on the osmotic stress resistance response from a molecular mechanism, and has important significance on the directed genetic improvement of alfalfa

Drawings

FIG. 1 is the response of the MsSAP22 gene to drought stress;

FIG. 2 is the cloning of the MsSAP22 gene;

FIG. 3 shows the growth of transformed yeast cells under stress of PEG and ABA;

FIG. 4 shows the construction of a plant overexpression vector for the MsSAP22 gene;

FIG. 5 is the subcellular localization of the MsSAP22 protein;

FIG. 6 is a PCR assay of MsSAP22 gene over-expression strains using hygromycin resistance gene primers;

FIG. 7 is the expression level of transgenic Arabidopsis MsSAP 22;

FIG. 8 is MsSAP22 transgenic Arabidopsis thaliana stress tolerance evaluation under Mannitol (Mannitol) and ABA stress;

fig. 9 is an assessment of MsSAP22 transgenic arabidopsis soil drought resistance.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. However, the specific experimental procedures referred to in the following examples were carried out in a conventional manner or under the conditions recommended by the manufacturer's instructions unless otherwise specified.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are all conventional methods unless otherwise specified. The reagents and materials used are commercially available, unless otherwise specified.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are exemplary only.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination, and bioinformatics, which will be apparent to those skilled in the art. These techniques are explained fully in the published literature, and the methods of DNA extraction, phylogenetic tree construction, gene editing method, gene editing vector construction, gene editing plant acquisition, and the like used in the present invention can be realized by methods already disclosed in the prior art, in addition to the methods used in the following examples.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, either single-stranded or double-stranded structures. These nucleic acids or polynucleotides include, but are not limited to, regulatory sequences for gene coding sequences, antisense sequences, and non-coding regions. These terms include a gene. "Gene" or "gene sequence" is used broadly to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons as in genomic sequences, and/or include coding sequences as in cDNA, and/or include cDNA and its regulatory sequences. In particular embodiments, e.g., with respect to an isolated nucleic acid sequence, it is preferred to default to cDNA.

"Expression vectors" refer to vectors in which Expression elements (such as promoter, RBS, terminator, etc.) are added on the basis of the basic skeleton of the cloning vector to enable the Expression of the target gene.

An Agrobacterium-mediated transformation method, agrobacterium-mediated transformation, refers to a technique of inserting a target gene into a modified T-DNA region, transferring and integrating an exogenous gene into a plant cell by virtue of Agrobacterium infection, and then regenerating a transgenic plant by cell and tissue culture techniques.

Example 1 real-time fluorescence quantification

MsSAP22 gene specific primers were designed using Primer 5.0 software, and the Primer sequences are shown in Table 1. The apparatus used for the experiment was a Bio-Rad CFX Maestro real-time fluorescent quantitative PCR system using a production 2 XSG Fast qPCR Master Mix kit, with 3 replicates per sample. The PCR reaction system is 10 mu L, and comprises 5 mu L of 2 XSG Fast qPCR Master Mix,1 mu L of DNF Buffer and 0.4 mu L of forward and reverse primers respectively; mu.L template cDNA, 2.2. Mu.L Sterilized ddH ₂ And O. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, followed by 40 cycles consisting of denaturation at 95 ℃ for 15s and annealing extension at 60 ℃ for 30s. With FC =2 ^-ΔΔCT The method calculates the relative expression level of a gene using MsActin as an internal reference gene (FIG. 1).

TABLE 1 qRT-PCR analysis primer List

EXAMPLE 2 cloning of the Gene

(1) Primer design

Designing specific primers (table 2) with enzyme cutting sites according to the genome reference sequence, wherein the primers are synthesized by Shanghai biological engineering GmbH;

TABLE 2 specific primer information for cloning MsSAP22

Note: the protecting bases are indicated in bold, and the enzyme sites are indicated underlined.

(2) Cloning of the fragment of interest

The PCR amplification reaction system is shown in the following table (Table 3):

TABLE 3 MsSAP22 cloning PCR reaction System

The PCR amplification reaction program is as follows:

2 μ L of PCR reaction product was loaded on a 2% agarose gel lane, and the target band was detected under a gel imager after electrophoresis at 135V and 400mA for 18 min. The PCR reaction product with the target fragment was collected in a 1.5mL centrifuge tube, and the PCR product was purified according to the instructions for purification of PCR products on the SanPrep column type, manufactured by Biotechnology engineering (Shanghai). After purification was complete, the concentration and quality of the purified product was determined using a NanoDrop ND 8000 ultramicro spectrophotometer for subsequent vector construction (fig. 2).

(3) Obtaining linear fragments

Carrying out double enzyme digestion on the purified fragment and pYES2 vectorThe reaction is carried out in an enzyme digestion system: 1. Mu.L Kpn I restriction enzyme, 1. Mu.L Xho I restriction enzyme, 2. Mu.L 10 XM buffer, 1. Mu.g plasmid template amount, ddH addition ₂ O is added to the volume of 20 mu L. The PCR reaction program is 37 ℃ and 3h;

(4) Gel recovery of vector fragments

Performing gel recovery on the linear vector fragment obtained by double enzyme digestion, wherein the gel recovery step refers to the specification of SanPrep column type DNA gel recovery (Shanghai Producer);

(5) Ligation of fragments of interest

And configuring an MsSAP22-pYES2 plasmid recombinant system in a sterile PCR tube, and then placing the mixture in a PCR instrument for reaction at 16 ℃ for 30min. The recombinant is: 1 u L T DNA connection buffer, 4 u L of target fragment DNA,2 u L linearized expression vector, 0.5 u L T DNA ligase, 2.5 u L ddH ₂ O；

And transforming the ligation product into escherichia coli competent DH5 alpha, culturing overnight, selecting a monoclonal for PCR detection, and detecting a proper extracted plasmid for later use.

Example 3 functional validation of heterologous expression by Yeast

1. Preparation of Yeast competent cells

The PEG/LiAc method is adopted to transform the yeast cells, and the specific steps are as follows:

(1) Streaking yeast strain stored at-80 deg.C on YPDA solid medium, 2-3 days later, picking single clone into 10ml LYPDA liquid medium (adding 10 μ L Kan), shaking culturing at 28 deg.C overnight to make OD ₆₀₀ ＝0.4；

(2) Yeast cells after overnight culture were diluted to OD in 50mL of medium ₆₀₀ ＝0.25；

(3) Yeast cells were incubated at 30 ℃ for about 4-5h until OD ₆₀₀ ＝1.0；

(4) Centrifuging the yeast cells at 2,500rpm for 5min, removing supernatant, and then resuspending the yeast cells with 40mL of 1 XTE;

(5) The suspension was centrifuged again at 2,500rpm for 5min, the supernatant was removed and the cells were resuspended in 2mL of 1 XLiAc/0.5 XTE buffer;

(6) Cells were revived at 28 ℃ for 10min.

2. Induction of Whole yeast transformants

(1) Add 1. Mu.g plasmid DNA, 100. Mu.g salmon sperm DNA (10 mg/mL) and 100. Mu.L resuspended yeast cells for each transformation, flick and mix;

(2) Adding again 700 μ L of 1 × LiAc/40% PEG-3350/1 × TE, mixing by gentle inversion;

(3) Incubating in 28 deg.C incubator for 30min;

(4) Adding 88 μ L dimethyl sulfoxide (DMSO), mixing by inversion, and performing heat shock at 42 deg.C for 7min;

(5) Centrifuging at 2500rpm for 15s, and removing supernatant;

(6) 1 × TE buffer solution of 1mL resuspends yeast cells, and centrifugates at a high speed to remove supernatant;

(7) Repeating the step 6 once;

(8) Resuspend yeast cells again in 1mL of 1 XTE buffer;

(9) Sucking 50 mu L of the heavy suspension liquid and coating the heavy suspension liquid on an SC-U-glucose culture medium;

(10) Culturing in 28 deg.C incubator for 1-2 days, and performing PCR detection on the monoclonal.

3. Verification of heterologous expression function of yeast

(1) Selecting a positive monoclonal containing a target gene and a monoclonal containing only pYES2 empty vector, inoculating the monoclonal to 10mL of SC-u/2% (w/v) glucose liquid culture medium with Amp resistance, and incubating the monoclonal for 20 hours at 28 ℃ and 200rpm in a shaking table;

(2) Measurement of OD of bacterial liquid ₆₀₀ The inoculum was inoculated into 10mL of SC-u/2% (w/v) galactose broth to obtain the OD of the new inoculum ₆₀₀ =0.4, shaking and culturing for 36h at 28 ℃ to promote exogenous gene expression;

(3) Sucking 200. Mu.L of the bacterial solution, and separating OD ₆₀₀ Adjusting to 1, performing instant centrifugation at 1000rpm for 10sec, discarding the supernatant, adding sterilized 30% (w/v) PEG-6000 solution, 250 μ M ABA solution and sterile water (control) in equal amount to the components simulating drought and ABA stress, and performing shake culture at 28 deg.C for 36h.

(4) Diluting the treated bacterial liquid by 10 times gradient (1, 10) ^-1 ，10 ^-2 ，10 ^-3 ，10 ^-4 ，10 ^-5 ，10 ^-6 ) 2 mul of the dilution was pipetted onto SC-u/2% (w/v) glucepinOn the plate, the plate was cultured in a 28 ℃ incubator for 3 to 4 days in a reversed manner, and the growth of colonies was observed and recorded by photographing (FIG. 3).

To further confirm the possible role and function of the MsSAP22 gene under drought stress, we performed an independent stress resistance test on the MsSAP22-pYES2 transformed yeast strain INVSc1 with the empty vector pYES2 as a control. The effect of overexpressing MsSAP22 on the survival of yeast cells in 30% peg and 250 μ M ABA medium was studied. As shown in FIG. 3, there was almost no difference in the growth of the MsSAP22 transgenic yeast cells from the empty vector control yeast cells under non-stress conditions, whereas the MsSAP22 transformed lines survived better under the action of 30% PEG and 250. Mu.M ABA, and the control no-load transformed yeast cells were strongly inhibited. These results indicate that MsSAP22 increases stress tolerance of yeast cells, and may have a strong effect on alfalfa response to abiotic stress.

Example 4 Gene location and functional identification

1. Plant expression vector construction

(1) Primer design

Designing specific primers (table 2) with enzyme cutting sites according to the genome reference sequence, wherein the primers are synthesized by Shanghai biological engineering GmbH;

(2) The cloning system of the target gene is shown in the following table (table 4):

table 4 pHG: : msSAP22 PCR cloning system

The PCR amplification reaction program is as follows:

the electrophoresis and purification method was the same as in example 2;

(3) Obtaining linear fragments

Carrying out double enzyme digestion reaction on the purified gene fragment and the pHG plasmid, wherein the system is as follows: mu.L of Pst I restriction enzyme1. Mu.L of Hind III restriction enzyme, 2. Mu.L of 10 XM buffer, 1. Mu.g of plasmid template, ddH ₂ O was brought to a constant volume of 20. Mu.L (FIG. 4).

2. Tobacco transient transformation

(1) Adding the agrobacterium tumefaciens monoclonal with positive detection into 10mL LB liquid culture medium with Kan resistance at 28 ℃, and performing overnight amplification at 200 rpm;

(2) Centrifuging 1.5mL of bacterial solution at room temperature of 8,000rpm for 2min, discarding the supernatant, and adding 1mL of penetrating fluid to suspend the thallus;

(3) Repeating the step (2) once;

(4) Measurement of bacterial liquid OD ₆₀₀ Value, resuspend the inoculum in 1.5mL centrifuge tube to make the OD of new inoculum ₆₀₀ Standing at room temperature for 2-3 hr to reach infection concentration of 0.4-0.6;

(5) Pouring enough water into the tobacco before infection, transferring the tobacco to a white fluorescent lamp 1h in advance to ensure that the air holes of the tobacco are fully opened to be favorable for injection, and selecting two leaves (inverted three leaves and inverted four leaves) for each plant to infect the same bacterial liquid;

(6) Gently erasing the lower epidermis wax at the back of the blade without the main vein by using a 1mL needle-free injector to form an injection point and mark a region to be transferred;

(7) Sucking 1mL of suspension by using a syringe without a needle, slightly pushing the suspension into an injection point, and stopping injection when the injection cannot be continuously diffused on the leaf back; four spots were injected per leaf, five plants were injected with gene and no-load. Marking the infected area with a marker pen;

(8) After the injection is finished, spraying water to the leaves, sleeving black plastic bags, carrying out dark culture in an incubator at 25 ℃ for 12 hours, and then transferring to normal culture under light.

3. Observation of target protein expression by fluorescence microscope

An MsSAP22-eGFP fusion protein under the control of a CaMV35S promoter is constructed and transiently expressed in nicotiana benthamiana epidermal cells, and an eGFP empty vector is used as a positive control. The tobacco leaves infected for two days are used as experimental materials and observed under a laser confocal microscope. Under confocal microscopy, the eGFP empty vector was uniformly distributed within the cells (fig. 5). The fluorescence signals of the MsSAP22-eGFP fusion protein are distributed in a dot shape and can be overlapped with the chloroplast autofluorescence signal, which indicates that the MsSAP22 expression protein is positioned in chloroplast (FIG. 5).

4. Arabidopsis transformation screening and positive plant detection

In order to detect whether the transgenic arabidopsis plants obtained by genetic transformation are positive plants, the research extracts total DNA from 16 obtained transgenic arabidopsis strains, and uses hygromycin resistance gene (598 bp) primers to carry out PCR detection, and the result shows that the DNA of all the strains can amplify specific bands, and the total positive rate reaches 100 percent (figure 6). After homozygous screening, the present study identified the expression levels of the MsSAP22 genes of five transgenic homozygous lines. The results show that the expression levels of the MsSAP22 genes in the five pure strains are obviously improved (figure 7), and OE4, OE9 and OE11 with the highest expression quantity are selected for subsequent drought resistance evaluation experiments.

5. Evaluation of stress resistance of transgenic Arabidopsis thaliana

In order to evaluate the drought resistance of the transgenic arabidopsis at the seedling stage, seeds are germinated on a non-resistant 1/2 MS culture medium, ten-day-old arabidopsis seedlings are paved on a 1/2 MS culture medium and a 1/2 MS culture medium containing 300mM Mannitol (Mannitol) and 30 mu M ABA, 3 biological repeats are set, the original positions of the root lengths of the arabidopsis seedlings are marked, the arabidopsis seedlings are vertically placed and cultured, the growth conditions of the root systems of the arabidopsis seedlings are observed after 7 days, the relative root lengths of the arabidopsis seedlings are measured and calculated to evaluate the drought resistance of the transgenic arabidopsis seedlings, and the result is shown in figure 8, and the growth of wild-type plants and transgenic plants has no significant difference on a 1/2 MS culture medium. On a 1/2 MS culture medium containing 300mM mannitol and 30 mu M ABA, the root growth of a wild type and a transgenic line is inhibited, but the root length and the lateral root number of the transgenic line are obviously higher than those of the wild type, which shows that the MsSAP22 gene can obviously improve the osmotic stress resistance of the transgenic Arabidopsis.

In addition to drought resistance evaluation on the medium, this study also evaluated the drought resistance of transgenic arabidopsis in soil. Firstly, col-0 and a transgenic strain are germinated on a normal 1/2 MS culture medium, and then the germinated arabidopsis seedlings are transplanted into soil, wherein the soil comprises turfy soil and vermiculite (3: 1). After the seedlings grow for about 20 days, the flower pots are watered until the seedlings are saturated, drought treatment is carried out, 3 biological repetitions are set for each treatment, then the phenotype of the seedlings is observed, and the results are shown in fig. 9. At the moment, all arabidopsis thaliana is subjected to rehydration treatment, and the result shows that the survival rate of the transgenic arabidopsis thaliana after rehydration is significantly larger than that of a wild type, so that the MsSAP22 enhances the drought resistance of the transgenic arabidopsis thaliana.

The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily made by those skilled in the art by replacing or changing the technical contents disclosed in the specification, and therefore, all changes and modifications that are made on the principle of the present invention should be included in the scope of the claims of the present invention.

Claims (8)

1. The alfalfa chloroplast MsSAP22 gene is characterized in that the nucleotide sequence of the MsSAP22 gene is shown in SEQ ID No. 1.

2. A protein encoding the MsSAP22 gene of claim 1, wherein the protein has an amino acid sequence as set forth in SEQ ID No. 2.

3. An overexpression vector comprising the alfalfa chloroplast MsSAP22 gene of claim 1.

4. Use of the gene of claim 1 or the protein of claim 2 or the overexpression vector of claim 3 for increasing drought resistance in a plant.

5. The use of claim 4, wherein the plant comprises alfalfa, tobacco, arabidopsis thaliana.

6. The use according to claim 4, wherein the drought tolerance is as follows: the tolerance of the transgenic plants is stronger than that of the wild type plants under osmotic stress resistance, relative to the wild type plants.

7. A plant breeding method, characterized in that the method is (1) or (2):

(1) Obtaining a plant with drought resistance stronger than that of a target plant by increasing the activity of MsSAP22 protein in the target plant;

(2) By promoting the expression of the MsSAP22 gene in the target plant, the plant with the drought resistance stronger than that of the target plant is obtained.

8. A method as claimed in claim 7, wherein the plant of interest comprises alfalfa, tobacco, arabidopsis thaliana.

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