CN116514941B - MsRGP1 protein, its encoding gene and its application in improving plant drought resistance and salt tolerance - Google Patents

Abstract

The application discloses MsRGP protein, a coding gene thereof and application thereof in improving drought resistance and salt tolerance of plants, wherein the amino acid sequence of MsRGP protein is shown as SEQ ID NO:1, the nucleotide sequence of MsRGP gene of the protein is shown as SEQ ID NO: 2. MsRGP1 genes are obtained by screening and separating alfalfa, and are found to simultaneously respond to salt, drought and ABA stress, and can regulate and control stress resistance of plants, such as salt tolerance, drought resistance and ABA stress resistance. The application also discloses application of the MsRGP protein, the coding gene, the recombinant vector or the recombinant bacterium carrying the recombinant vector in improving drought resistance and/or salt tolerance of plants. The MsRGP protein and the coding gene thereof can be applied to the cultivation of transgenic plants, wherein the transgenic plants are plants with improved salt stress tolerance and drought resistance or plants with reduced salt stress tolerance and drought resistance, and can be used as new materials for actual production or scientific research application, thereby playing a role.

Description

MsRGP1 protein, its encoding gene and its application in improving plant drought resistance and salt tolerance

Technical Field

The invention relates to the technical field of genetic engineering, in particular to MsRGP1 protein, its coding gene and its application in improving plant drought resistance and salt tolerance.

Background technique

Land salinization and drought have always been important factors restricting agricultural development. About 20% of the world's irrigated land is affected by land salinization, and about 40% of the land is affected by drought. On saline-alkali and arid land, plants face multiple unfavorable factors such as water shortage, high salt and high pH. Biomass accumulation and growth rate are significantly reduced, the ability of the root system to absorb water and grow downward is limited, and the osmotic balance and ion balance are unbalanced, which ultimately causes plant metabolic disorders, yellowing of leaves, withering and even death. In my country, the area of saline-alkali land and arid land has increased year by year, reducing crop yields and restricting agricultural development.

Therefore, how to alleviate land salinization, improve land and restore land productivity is an important issue related to the development of my country's agriculture. Among them, the development of plants with excellent stress resistance is particularly important. It can be used as one of the crops for improving land and restoring land productivity, among which alfalfa is a typical representative.

Alfalfa ( Medicago sativa ) is an autotetraploid, cross-pollinated plant of the genus Medicago in the Leguminosae family. It has the advantages of biological nitrogen fixation, high protein content, good quality, and a wide range of planting, and is known as the "king of forage grasses." However, as the global climate and environmental situation becomes increasingly severe, the growth of alfalfa is affected by extreme environments, including abiotic stresses such as salinity and drought, resulting in reduced yield and quality. Using molecular biological methods to improve the tolerance of alfalfa to salinity and drought stress is an effective way to increase alfalfa yield and improve land.

Therefore, it is of great significance to discover new genes related to response to salinity and drought stress and to cultivate new plant materials with stronger stress tolerance.

Therefore, the existing technology needs to be further developed.

Summary of the invention

In response to the above technical problems, the present invention provides a MsRGP1 protein isolated from alfalfa, its encoding gene MsRGP1 gene and its application in improving plant drought resistance and salt tolerance. Downregulation of the expression of the gene in plants can improve the drought resistance and salt tolerance of the plants, which is helpful for cultivating new stress-resistant plant varieties.

To solve the above problems, the applicant of the present invention analyzed the expression patterns of members of the MsRGP gene family of alfalfa under salt, drought and ABA stress, combined with transcriptome data, and finally screened out MsRGP1 that responds to salt, drought and ABA stress. By analyzing plants overexpressing MsRGP1 and mutants of the gene, the gene function of MsRGP1 in salt-alkali, drought and ABA stress was verified in Arabidopsis thaliana and alfalfa, and it was determined that the gene negatively regulates the plant's salt-alkali and drought resistance, and positively regulates the plant's resistance to ABA stress.

Specifically, the technical solutions provided by this application are as follows:

First, the present application provides a MsRGP1 protein, whose amino acid sequence is shown in SEQ ID NO: 1.

The above amino acid sequence includes the sequence shown in SEQ ID NO: 1, and also includes homologous sequences with 95% or more identity to the protein sequence, which can be obtained by mutating the sequence shown in SEQ ID NO: 1 using existing technology. The protein activity of these homologous sequences is the same as the protein activity of SEQ ID NO: 1.

In order to facilitate purification or detection of the above protein, a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 1.

The tag protein includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein and other tag proteins.

In a second aspect, the present application provides the MsRGP1 gene encoding the above protein, and its nucleotide sequence is shown in SEQ ID NO:2.

The above gene sequence includes not only the sequence shown in SEQ ID NO: 2, but also homologous sequences with 95% or more identity to the gene sequence, which can be obtained by mutating the sequence shown in SEQ ID NO: 2 using existing technology. The protein activity expressed by these homologous sequences is the same as the protein activity expressed by the sequence of SEQ ID NO: 2.

In addition, those artificially modified nucleotides that have 95% or more identity with the nucleotide sequence encoding the protein MsRGP1 isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as the function and activity of the encoded protein are basically the same.

The present application screened the MsRGP1 gene that responds to salt, drought and ABA stress from alfalfa. On the one hand, an overexpression vector pFGC-MsRGP1-eYFP was constructed, and Arabidopsis thaliana was transformed using the Agrobacterium-mediated method. After screening and identification, MsRGP1 transgenic Arabidopsis thaliana strains were obtained; on the other hand, the MsRGP1 gene knockout strain of Arabidopsis thaliana was studied at the same time.

Through the observation of Arabidopsis seed germination and seedling growth under salinity, drought and ABA stress, it was found that compared with the wild type, overexpression of MsRGP1 reduced Arabidopsis tolerance to salinity and drought, and increased tolerance to exogenous ABA stress; while Arabidopsis mutants had higher tolerance to salinity and drought than the wild type, and lower tolerance to exogenous ABA stress than the wild type. Through the observation of Arabidopsis seedling under drought stress, it was found that overexpression of MsRGP1 reduced Arabidopsis tolerance to drought, while Arabidopsis mutants had higher tolerance to drought than the wild type.

In addition, the applicant found through research on alfalfa overexpressing MsRGP1 that under salt treatment and drought treatment, overexpression of MsRGP1 reduced the tolerance of alfalfa to salt and drought stress, that is, the salt tolerance and drought tolerance of MsRGP1 overexpressing alfalfa were lower than those of the wild type, which indicates that overexpression of MsRGP1 reduced the tolerance of alfalfa to salt and drought stress. Therefore, the expression of MsRGP1 can be reduced through gene editing to improve the tolerance of alfalfa to salt and drought stress, and obtain new alfalfa materials with better salt and drought resistance. This technical effect is foreseeable.

Therefore, the MsRGP1 gene is a new important gene that affects the salt-alkali and drought resistance of alfalfa and Arabidopsis thaliana. It can be used to breed new plant materials with stronger stress resistance. The application value of this gene is high.

In a third aspect, the present application provides a recombinant vector carrying the aforementioned MsRGP1 gene or the aforementioned MsRGP1 protein.

The vectors described herein are well known to those skilled in the art, including but not limited to: plasmids, bacteriophages, cosmids (i.e., cosmids), Ti plasmids or viral vectors. The plasmids are existing expression vectors such as pFGC or existing cloning vectors such as pMD19-T vectors, and are not limited to the specific types of the embodiments of the present application.

Existing plant expression vectors can be used to construct a recombinant expression vector containing the MsRGP1 gene. When the MsRGP1 gene is used to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before the transcription start nucleotide.

In addition, when constructing a plant expression vector using the gene of the present invention, an enhancer may also be used, including a translation enhancer or a transcription enhancer, and the enhancer region may be an ATG start codon or an adjacent region start codon, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are wide, and may be natural or synthetic.

In a fourth aspect, the present application provides a recombinant bacterium, which includes the aforementioned recombinant vector.

The recombinant bacteria herein may be bacteria, fungi, yeast or algae. Among them, the bacteria may be from Escherichia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc. Specifically, it may be Escherichia coli DH5α and/or Agrobacterium tumefaciens EHA105.

In a fifth aspect, the present application also provides the use of the aforementioned MsRGP1 protein, its encoding gene, the aforementioned recombinant vector or a recombinant bacterium carrying the recombinant vector in improving plant drought resistance and/or salt tolerance.

Based on the aforementioned functions and effects of MsRGP1, those skilled in the art can apply the aforementioned MsRGP1 protein and its encoding gene to cultivate MsRGP1 transgenic plants based on conventional experimental methods and existing technologies in the art. The transgenic plants are plants with improved salt stress tolerance and drought resistance or plants with reduced salt stress tolerance and drought resistance, and can be used as new materials for actual production or scientific research applications, thereby playing a role.

Preferably, the plant is alfalfa or Arabidopsis thaliana.

Preferably, in the aforementioned application of improving plant drought resistance and/or salt tolerance, the expression level of MsRGP1 gene is down-regulated or the MsRGP1 gene is knocked out in the target plant to improve the drought resistance and salt tolerance of the plant, thereby obtaining a superior new variety.

In a sixth aspect, the present application also provides the use of the aforementioned MsRGP1 protein, its encoding gene, the aforementioned recombinant vector or a recombinant bacterium carrying the recombinant vector in improving the tolerance of plants to exogenous abscisic acid stress. Optionally, the plant is Arabidopsis thaliana.

Experiments have shown that overexpression of MsRGP1 improves Arabidopsis' tolerance to exogenous abscisic acid (ABA) stress compared to the wild type.

Preferably, in the application of improving the tolerance of plants to exogenous abscisic acid stress, the tolerance of plants to exogenous abscisic acid stress is improved by up-regulating the expression level of MsRGP1 gene in the plants.

In a seventh aspect, the present application also provides a method for preparing a transgenic plant, the method being the following (1) or (2):

(1) By reducing the overexpression of the MsRGP1 gene in the target plant, a plant with stronger salt stress tolerance and drought resistance than the target plant is obtained;

(2) By promoting the expression of the MsRGP1 gene in the target plant, plants with lower salt stress tolerance and drought resistance than the target plant are obtained.

In the above preparation method, the method for reducing the expression level of the MsRGP1 gene in the target plant is to use gene mutation, gene knockout, gene editing or gene knockdown techniques to reduce or inactivate the activity of the MsRGP1 gene in the genome of the target plant, thereby reducing the effect of MsRGP1 expression, thereby obtaining plants with stronger salt stress tolerance and drought resistance than the target plant, and using them as dominant plants.

The above-mentioned promotion of the expression of the MsRGP1 gene in the target plant can be achieved by utilizing gene overexpression technology (such as introducing an expression vector carrying the encoding gene into the target plant) to increase the expression level of MsRGP1 in the plant.

In the above method, the plant may be a plant with altered stress resistance. Further, the plant with altered stress resistance may be a transgenic plant with reduced (downregulated) and/or improved (upregulated) stress resistance (such as salt tolerance and/or drought resistance).

The MsRGP1 protein, the encoding gene thereof and the application thereof in improving the drought resistance and salt tolerance of plants provided by the present invention have the following beneficial effects:

1. The present invention screened the MsRGP1 gene from the genome of alfalfa ( Medicago sativa ) for the first time, and found that it responds to salt, drought and ABA stress simultaneously. By introducing the gene into the recipient plants Arabidopsis and alfalfa, transgenic Arabidopsis and transgenic alfalfa overexpressing the MsRGP1 gene were obtained, and the salt-alkali resistance, drought resistance and ABA stress resistance of these transgenic plants and the MsRGP1 gene mutant of Arabidopsis were analyzed and identified. Based on the results of various physiological and biochemical index measurements, it was found that compared with wild-type plants, overexpression of MsRGP1 reduced the tolerance of Arabidopsis and alfalfa to salt-alkali and drought, and improved the tolerance of Arabidopsis to exogenous ABA stress; while the tolerance of Arabidopsis mutants to salt-alkali and drought was higher than that of the wild type, and the tolerance to exogenous ABA stress was lower than that of the wild type.

2. The MsRGP1 protein and the encoding gene MsRGP1 of the present invention can regulate the stress resistance of plants (such as salt tolerance, drought resistance, and resistance to ABA stress), and can cultivate high-quality plants that are salt-tolerant and/or drought-resistant by reducing the content and/or activity of the protein in the target plant (such as inhibiting the expression of the gene). The MsRGP1 and the MsRGP1 encoding gene provided by the present invention have important theoretical significance and application value in regulating the salt and drought resistance of Arabidopsis and alfalfa, and in cultivating superior stress-resistant varieties.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the expression pattern analysis of MsRGP gene family members under salt stress; Note: 0 d: before treatment; 1 d: first day of salt treatment; 6 d: sixth day of salt treatment;

FIG2 is an analysis of the expression pattern of the MsRGP gene family in roots, stems and leaves of alfalfa;

FIG3 is a heat map of the expression of MsRGP gene family members under drought and ABA stress;

FIG4 is a graph showing the RNA integrity test of alfalfa;

FIG5 is the result of amplification of the MsRGP1 gene fragment;

Figure 6 shows the identification of pFGC- MsRGP1 -eYFP vector; Note: A: vector identification result; M: Marker DL2000; 1-5: target bands; B: pFGC-MsRGP1-eYFP vector;

FIG7 is the result of amino acid sequence alignment between MsRGP1 and MtRGP1;

Figure 8 shows the screening of resistant Arabidopsis plants. Note: The red circles indicate that the surviving Arabidopsis plants may be positive plants.

FIG. 9 shows the identification results of positive plants of MsRGP1 transgenic Arabidopsis thaliana; M: Marker DL2000; WT: wild-type Arabidopsis thaliana; H ₂ O: water; Numbers: different numbers represent different strains.

FIG. 10 is the verification of the expression level of MsRGP1 transgenic Arabidopsis positive plants; the horizontal axis is the serial number of Arabidopsis positive plants, and the vertical axis is the expression level.

Figure 11 shows the identification of homozygous mutants of Arabidopsis thaliana; A: identification of homozygous mutants of atrgp1 Arabidopsis thaliana; B: identification of homozygous mutants of atrgp2 Arabidopsis thaliana; M: Marker DL2000; WT: wild-type Arabidopsis thaliana; 1 or 2: different strains; G: identification of gene sequence integrity; T: identification of T-DNA insertion.

Figure 12 shows the germination of wild-type Arabidopsis, MsRGP1 transgenic Arabidopsis and mutants under salt stress; A: germination of 5 strains under 0 mM NaCl treatment; B: germination of 5 strains under 125 mM NaCl treatment; C: germination of 5 strains under 150 mM NaCl treatment; D: germination rate line graph of 5 strains under 0 mM NaCl treatment; E: germination rate line graph of 5 strains under 125 mM NaCl treatment; F: germination rate line graph of 5 strains under 150 mM NaCl treatment;

Figure 13 shows the survival of wild-type Arabidopsis, MsRGP1 transgenic Arabidopsis and mutants under salt stress; Notes: A: survival of 5 strains under 0 mM NaCl treatment; B: survival of 5 strains under 125 mM NaCl treatment; C: germination of 5 strains under 150 mM NaCl treatment; D: survival rate of 5 strains under 0 mM NaCl treatment and 125 mM NaCl treatment; E: survival rate of 5 strains under 0 mM NaCl treatment and 150 mM NaCl treatment;

Figure 14 shows the germination of wild-type Arabidopsis, MsRGP1 transgenic Arabidopsis and mutants under drought stress; A: germination of 5 strains under 0 mM mannitol treatment; B: germination of 5 strains under 300 mM mannitol treatment; C: germination of 5 strains under 400 mM mannitol treatment; D: germination rate line graph of 5 strains under 0 mM mannitol treatment; E: germination rate line graph of 5 strains under 300 mM mannitol treatment; F: germination rate line graph of 5 strains under 400 mM mannitol treatment;

Figure 15 shows the survival of wild-type Arabidopsis, MsRGP1 transgenic Arabidopsis and mutants under drought stress; A: survival of 5 strains under 0 mM mannitol treatment; B: survival of 5 strains under 300 mM mannitol treatment; C: survival of 5 strains under 400 mM mannitol treatment; D: survival rate of 5 strains under 0 mM mannitol treatment and 300 mM mannitol treatment; E: survival rate of 5 strains under 0 mM mannitol treatment and 400 mM mannitol treatment;

Figure 16 shows the phenotypic analysis of wild-type Arabidopsis, MsRGP1 transgenic Arabidopsis and mutants under ABA stress; A: germination of wild-type Arabidopsis and MsRGP1 overexpressing Arabidopsis under 0 μM ABA treatment and 1 μM ABA treatment; B: germination of wild-type Arabidopsis and mutants under 0 μM ABA treatment and 1 μM ABA treatment; C: germination rate of wild-type Arabidopsis and MsRGP1 overexpressing Arabidopsis under 0 μM ABA treatment; D: germination rate of wild-type Arabidopsis and MsRGP1 overexpressing Arabidopsis under 1 μM ABA treatment; E: germination rate of wild-type Arabidopsis and mutants under 0 μM ABA treatment; F: germination rate of wild-type Arabidopsis and mutants under 1 μM ABA treatment; G: germination rate of wild-type Arabidopsis and MsRGP1 overexpressing Arabidopsis at 10th day under 0 μM ABA treatment and 1 μM ABA treatment. d green leaf situation; H: green leaf situation of wild-type Arabidopsis and mutants under 0 μM ABA treatment and 1 μM ABA treatment on the 20th day; I: green leaf rate of wild-type Arabidopsis and MsRGP1 -overexpressing Arabidopsis under 0 μM ABA treatment and 1 μM ABA treatment on the 10th day; J: green leaf rate of wild-type Arabidopsis and mutants under 0 μM ABA treatment and 1 μM ABA treatment on the 20th day;

Figure 17 shows the effects of abiotic stress on Arabidopsis seedlings; A: growth of seedlings of 5 strains under different treatments; B: root length of 5 strains after growth under different treatments; C: fresh weight of 5 strains after growth under different treatments.

FIG18 shows the effect of drought stress on Arabidopsis thaliana;

FIG19 shows the survival rate of Arabidopsis thaliana after drought treatment; Control: before treatment; Drought: after drought treatment;

FIG20 is an analysis of the expression level of MsRGP1 transgenic alfalfa positive plants;

Figure 21 is the phenotypic analysis of MsRGP1 transgenic alfalfa and wild-type alfalfa under salt stress; A: before treatment; B: 10 days of salt treatment; C: 15 days of salt treatment; D: 20 days of salt treatment;

Figure 22 is the phenotypic analysis of MsRGP1 transgenic alfalfa and wild-type alfalfa under drought stress; A: before treatment; B: 15 days of drought treatment; C: 17 days of drought treatment; D: 20 days of drought treatment;

The results are expressed as mean ± standard deviation (SD), and t- test was used for significance analysis ("*" P < 0.05, "**" P < 0.01).

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

Example 1 Screening of MsRGP1 gene

In order to better explore the salt-alkali tolerance genes of alfalfa, alfalfa was treated with different concentrations of salt, alkali and salt-alkali mixture in the early stage of the experiment, and transcriptome sequencing was performed on the different treatment groups of alfalfa. Through transcriptome data analysis, it was found that the MsRGP1 gene responded to salt-alkali stress, thus obtaining a new gene MsRGP1 that affects the salt-alkali resistance of alfalfa. The results of transcriptome sequencing analysis showed that the nucleotide sequence of the gene is shown in SEQ ID NO: 2, and the amino acid sequence of the protein expressed by the sequence is shown in SEQ ID NO: 1.

To study the MsRGP gene family, bioinformatics analysis and expression pattern analysis in different tissues and under various stresses were performed to predict and further verify the gene function of the gene.

1.1 Experimental methods

1.1.1 Stress treatment of alfalfa

Alfalfa seeds were placed in a culture dish covered with filter paper and kept moist. After 5 days of dark culture, the germinated seedlings were transferred to Hoagland nutrient solution for hydroponics, and the nutrient solution was replaced every 3 days. The culture conditions were 16 h light/8 h dark, the day and night temperature was 25°C/22°C, and the relative humidity was 60-70%. 30-day-old alfalfa with the same growth was selected for treatment. A control group (Hoagland nutrient solution) and a treatment group (salt, mannitol and ABA) were set up with three replicates. The treatment methods and sampling time were as follows:

Salt stress: The plants were treated with Hoagland's nutrient solution containing 100 mM NaCl, and samples were taken at 0 h, 3 h, 6 h, 12 h, 24 h and on the sixth day of treatment.

(2) Osmotic stress: The plants were treated with Hoagland nutrient solution containing 300 mM Manntiol, and samples were taken at 0 h, 3 h, 6 h, 12 h, 24 h and 2 d after treatment.

(3) ABA stress: The plants were treated with Hoagland nutrient solution containing 10 μM ABA, and samples were taken at 0 h, 3 h, 6 h, 12 h, and 24 h after treatment. The sampling sites were roots, stems, and leaves. All samples were quickly frozen with liquid nitrogen and stored in a −80 °C refrigerator until use.

1.1.2 qRT-PCR analysis of the MsRGP gene family

A more comprehensive expression pattern analysis of the MsRGP gene family was performed by qRT-PCR (Schmittgen et al., 2008). Specific primers for qRT-PCR were designed using Primer Premier 5 software (Table 1). RNA from alfalfa was extracted and reverse transcribed according to the instructions. qRT-PCR experiments were performed according to the instructions. Three replicates were set for each sample, and the data were analyzed using the 2−∆∆CT method. The results are expressed as mean ± standard deviation (SD). The t-test was used to calculate significant differences (“*” represents P < 0.05, “**” represents P < 0.01). The expression levels under drought and ABA stress are presented in the form of heat maps.

Table 1 qRT-PCR primers

1.2 Experimental Results and Analysis

1.2.1 Analysis of the expression pattern of MsRGP gene family under salt stress

(1) The expression patterns of MsRGP gene family members before and after 100 mM NaCl stress were analyzed using transcriptome data (Figure 1). The expression levels of MsRGP1 , MsRGP2 , and MsRGP5 showed an up-regulated trend on the first and sixth days under salt stress, with similar expression patterns. The expression level of MsRGP3 showed a down-regulated trend on the first day under salt stress and an up-regulated trend on the sixth day. Both MsRGP4 and MsRGP4a showed a down-regulated trend on the first and sixth days under salt stress. This indicates that the MsRGP gene family responded to salt stress.

(2) qRT-PCR analysis of the MsRGP gene family

As shown in Figure 2, the expression levels of MsRGP1 and MsRGP2 in roots and stems were higher than those of other family members. MsRGP5 had the highest expression level in leaves, followed by MsRGP1 . Overall, the expression level of MsRGP1 was higher than that of other MsRGP gene family members in roots, stems or leaves .

1.2.2 Analysis of expression patterns of MsRGP gene family members under drought and ABA stress

Under drought stress, the expression patterns of MsRGP1 and MsRGP2 were basically the same, with their expression levels downregulated in roots and stems, but upregulated in leaves. The expression levels of MsRGP4 in roots, stems and leaves were upregulated to varying degrees. In addition, the expression levels of MsRGP3 and MsRGP5 were upregulated in leaves (Figure 3A).

Under ABA stress, except for the expression of MsRGP2 in roots, the expression levels of the five MsRGP gene family members in roots, stems and leaves showed a trend of first increasing and then decreasing from 3 h to 12 h. Among them, the expression level of MsRGP1 in roots, stems and leaves was the highest at 3 h after ABA stress treatment (Figure 3).

The above results show that MsRGP1 and MsRGP2 sequences have similar expression patterns under salt and drought stress, and it is speculated that MsRGP1 and MsRGP2 may have certain gene redundancy in responding to salt and drought stress. By analyzing the transcriptome data under saline-alkali stress, it was found that only the MsRGP1 gene was differentially expressed. Therefore, it is speculated that MsRGP1 may be involved in the response to salt, drought and ABA stress. Therefore, MsRGP1 was selected for subsequent verification and detection of gene function.

Example 2 Preparation of Arabidopsis thaliana overexpressing MsRGP1 gene

2.1 Construction of MsRGP1 gene overexpression vector

To verify the gene function of MsRGP1 , the coding region (CDS) sequence of MsRGP1 was cloned in alfalfa and the pFGC- MsRGP1 -eYFP overexpression vector was constructed.

2.1.1 Experimental methods

(1) Amplification of the MsRGP1 coding region sequence

Extract RNA from SY4D of alfalfa. Refer to the instructions for specific steps (FastPure Universal PlantTotal RNA Isolation Kit). Reverse transcribe the RNA to obtain the cDNA product of alfalfa. Design primers using PrimerPrimer 5.0 software (Table 2), use the primers to amplify and sequence MsRGP1 , and select the correct product by comparison to obtain the target fragment.

Table 2 Primer information

(2) Construction of overexpression vector

Use Bam HⅠ restriction endonuclease to perform single restriction digestion on the plasmid of the pFGC-eYFP overexpression vector. Use a seamless cloning kit to connect the target fragment to the overexpression vector after restriction digestion. The specific steps are carried out according to the instructions. After the ligation reaction is completed, the ligation product is transferred into DH5a chemical competent cells. After the transformation is completed, the bacterial solution is spread on LB medium containing Kan (50 mg/L) and inverted in a 37°C incubator for 24~36 h. Select a single clone for bacterial solution PCR identification and sequencing, and use primers as shown in Table 2. Activate and preserve the bacterial solution with correct sequencing. Extract the plasmid of the MsRGP1 overexpression vector and transfer it into EHA105 Agrobacterium competent cells. The specific steps are as follows:

① Take the competent Agrobacterium stored at −80℃ and place it at room temperature or in the palm of your hand for a while to partially melt it. When it is in a state of ice-water mixing, insert it into ice.

② Add 0.01-1 μg plasmid DNA per 100 μl competent cell, gently stir the bottom of the tube to mix, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

③ Add 700 μl of LB liquid culture medium and culture at 28℃ with shaking for 2-3 h.

④ Centrifuge at 6000 rpm for 1 min to collect the bacteria, take about 100 μl of the supernatant, gently blow and resuspend the bacteria, spread it on LB medium containing Kan antibiotics (50 mg/L), and place it upside down in a 28℃ incubator for 2-3 days

⑤ Perform PCR identification on the bacterial liquid of the grown single clone.

The pFGC- MsRGP1 -eYFP overexpressing Agrobacterium culture medium was added to LB liquid medium containing Kan (50 mg/L) and Rif (50 mg/L) for activation at 28°C. After 36-48 h, when the culture solution became turbid, it was mixed with glycerol in a 1:1 ratio for bacterial preservation, frozen with liquid nitrogen, and stored at −80°C.

(3) Infection and subcellular localization of tobacco leaves

The pFGC- MsRGP1 -eYFP Agrobacterium culture liquid and the pFGC-eYFP empty Agrobacterium culture liquid constructed above were activated in LB liquid culture medium containing Rif and Kan until the _OD600 of the culture liquid reached between 0.5 and 1.5. The supernatant was discarded after centrifugation, and the cells were resuspended in 10 mM _MgCl2 suspension containing 120 μM AS until the _OD600 of the resuspended liquid was about 0.6. The suspension was cultured in the dark at 25°C for 2 h.

Select one-month-old tobacco plants with good growth, use a 1 mL syringe to absorb the resuspension and inject it into the lower epidermis of the tobacco leaves and mark it. After the infection is completed, the infected tobacco is treated in the dark at 25℃ for 10-14 hours and then restored to the normal light cycle for 2-3 days. After that, the tobacco leaves marked with the injection position are sampled and made into slides and photographed for record.

2.1.2 Experimental results and analysis

(1) Cloning of CDS of MsRGP1

The extracted alfalfa RNA was tested for quality. The concentration of the extracted alfalfa RNA was about 200 ng/μl. After gel electrophoresis, two clear bands were detected, indicating that the RNA was not degraded and could be used for subsequent experiments (Figure 4).

Using alfalfa cDNA as a template, the MsRGP1 gene CDS was amplified and the PCR product was detected by gel electrophoresis. The band size was consistent with the expected result (Figure 5).

The sequencing results showed that the CDS length of the MsRGP1 gene was 1074 bp, which was consistent with the sequence obtained from the transcriptome, indicating that the CDS of MsRGP1 had been successfully cloned. The CDS sequence of MsRGP1 was translated into an amino acid sequence and compared with the amino acid sequence of MtRGP1 of Medicago truncatula. The results showed that the amino acid sequence similarity between MsRGP1 and MtRGP1 was about 98.61% (Figure 7).

(2) Construction of MsRGP1 overexpression vector

The CDS of the amplified MsRGP1 gene was connected to the pFGC-eYFP vector by seamless cloning and PCR verification. The result showed that the vector was successfully connected (Figure 6). The recombinant vector was named pFGC- MsRGP1 -eYFP. The promoter of this vector is cauliflower mosaic virus 35S (CaMV35S), with eYFP marker gene, and has Kan and PPT resistance.

This example uses the overexpression vector pFGC- MsRGP1 -eYFP with the promoter CaMV35S for subcellular localization. The strong CaMV35S promoter can increase the expression level of the YFP fusion protein without affecting the subcellular localization results. Subcellular localization of the MsRGP1 protein in tobacco leaves showed that the MsRGP1 protein was localized in the cytoplasm and nucleus.

2.2 Obtaining MsRGP1 transgenic Arabidopsis

2.2.1 Experimental methods

(1) Arabidopsis thaliana cultivation

Take an appropriate amount of wild-type Arabidopsis seeds in a 1.5 mL sterilized centrifuge tube, wash with dd H ₂ O, and then sterilize in 75% alcohol for 1-2 min; wash again with dd H ₂ O; soak in 10% NaClO solution for 7-10 min; wash 5-8 times with dd H ₂ O and let stand for 30 min. Spread Arabidopsis seeds evenly on 1/2MS medium; after vernalization at 4℃ for 2-3 days, place in a 24℃ light incubator for culture, and move to soil (vermiculite: nutrient soil = 20:1) after growing 4 true leaves.

(2) Infection of Arabidopsis thaliana by flower dipping method

Take out Agrobacterium transformed with MsRGP1 overexpression vector, add 100 μL of the bacterial solution to 10 mL of liquid LB medium containing Kan (50 mg/L) and Rif (50 mg/L), and culture it in a 28℃ shaker at 200 rpm for 2~3 days; add 1~2mL of activated bacterial solution to 200 mL of liquid LB medium containing Kan (50 mg/L) and Rif (50 mg/L), and shake it in a 28℃ shaker at 200 rpm until the bacterial solution _OD600 is 1.2~2.0; centrifuge at 6500 rpm for 15 min at 4℃, and discard the supernatant. Resuspend it with 5% sucrose solution to _OD600 of 1; activate the resuspension at 28℃ for 30 min; add 0.03% activator sliwet-77; immerse Arabidopsis inflorescence in the bacterial solution for 15~30 sec, culture it in the dark for one day, and then culture it normally; and perform the second infection after 7~8 days.

(3) Screening and identification of positive plants

After Arabidopsis thaliana was infected, mature seeds were harvested; after being sterilized, the seeds were evenly spread on 1/2MS medium containing PPT (7.5 mg/L) and cultured at 24°C in the light after two days of vernalization; seedlings with four true leaves were transferred to soil (vermiculite: nutrient soil = 20:1).

Arabidopsis DNA was extracted and PCR was performed for verification. The primer information is shown in Table 3. RNA of positive plants was extracted and reverse transcribed before qRT-PCR for expression analysis. The primer information is shown in Table 4-1. The 2-3 strains with the highest expression were selected, propagated, and _T3 generation seeds were harvested for subsequent experiments.

Table 3 Primer information

2.2.1 Results and analysis

The pFGC- MsRGP1 -YFP overexpression vector was transferred into Arabidopsis, and resistant plants were screened using 1/2MS medium containing PPT (Figure 8). Specific primers 35S-F and MsRGP1-R were used to identify the resistant plants. Seven positive lines of MsRGP1 transgenic Arabidopsis were obtained through DNA level identification (Figure 9). The expression levels of these seven positive lines were detected by qRT-PCR, and the two lines with the highest expression levels (OE17 and OE18) were selected for subsequent experiments (Figure 10).

Example 3 Screening of Arabidopsis mutants

3.1 Methods:

The Arabidopsis homologous genes AtRGP1 and AtRGP2 of MsRGP1 were found through the Tair online website (https://www.Arabidopsis.org/index.jsp), and the gene numbers are At3g02230 and At5g15650, respectively. The mutant strains of the two genes atrgp1 (N664336) and atrgp2 (N656992) were found on the Arashare online website (https://www.arashare.cn/index/). Arabidopsis mutant DNA was extracted for identification.

The identification method was double primer identification: N664336 was amplified by PCR using AtRGP1-F and AtRGP1-R, T-DNA-F and AtRGP1-R. When the former had no band and the latter had a band with the correct band size, it was a homozygous mutant. N656992 was amplified by PCR using AtRGP2-F and AtRGP2-R, T-DNA-F and AtRGP2-R, using the same method as above (see Table 3).

3.2 Results and Analysis

In order to verify the gene function of MsRGP1 in both directions , the mutant lines of MsRGP1 homologous genes AtRGP1 and AtRGP2 in Arabidopsis thaliana were screened. PCR identification of atrgp1 and atrgp2 was performed , and finally the homozygous mutant lines of atrgp1 and atrgp2 Arabidopsis thaliana were obtained (see Figure 11).

Example 4 Stress treatment methods and phenotypic analysis of transgenic and mutant Arabidopsis

4.1 Experimental methods

The following experiments were conducted using the MsRGP1 overexpression strains and two mutant strains obtained in Examples 2 and 3 as materials:

(1) Evaluation method of the effects of salinity, drought and ABA stress on seed germination

Wild-type Arabidopsis seeds, two MsRGP1 overexpression lines and two mutant lines were used as materials for salt treatment (125 mM and 150 mM NaCl), alkali treatment (5 mM NaHCO3), drought treatment (300 mM and 400 mM mannitol), ABA treatment (1 μM ABA) and blank control, with three biological replicates in each group. The seeds were cultured in a 24℃ light incubator, and the germination rate, survival rate and green leaf rate were biologically counted, and the phenotypic differences were observed and recorded.

(2) Evaluation method of the effects of salinity, drought and ABA stress on root length development

Wild-type Arabidopsis, two MsRGP1 overexpression lines and two mutant lines that had the same growth after germination for 7 days were used as materials. Salt treatment (125 mM NaCl), alkali treatment (5 mM NaHCO3), drought treatment (300 mM mannitol) and ABA treatment (30 μM ABA) and blank control were carried out, with three biological replicates in each group. The seeds were moved to a 24℃ light incubator for vertical cultivation. The root length was biologically counted 12 days after seed germination, and the phenotypic differences were observed and recorded.

(3) Evaluation method of the effect of drought stress on Arabidopsis seedling growth

After culturing wild-type Arabidopsis seeds, two MsRGP1 overexpression lines, and two mutant lines in 1/2MS medium for 7 days, the germinated Arabidopsis were transferred to vermiculite. After culturing in vermiculite for about 14 days, Arabidopsis was treated, including blank control and drought treatment (natural drought), with no less than 3 biological replicates for each treatment group. The drought treatment method was to stop watering until phenotypic differences or wilting occurred, rewater when wilting occurred, and then observe the survival phenomenon and perform biological statistics.

4.2 Results and Analysis

4.2.1 Effects of saline-alkali, drought and ABA stress on Arabidopsis seed germination

(1) Effects of salt stress on Arabidopsis seed germination

MsRGP1 transgenic Arabidopsis (OE17 and OE18), Arabidopsis mutants ( atrgp1 and atrgp2 ) and wild-type Arabidopsis were cultured on 1/2MS medium containing NaCl (125 mM and 150 mM). The results showed that:

Under 125 mM and 150 mM NaCl treatments, the germination rates of OE17 and OE18 were significantly lower than that of WT, especially at 2-4 d. The germination rates of atrgp1 and atrgp2 were significantly higher than that of WT ( P < 0.05) (Figure 12). The survival rates of the five strains were biologically statistically analyzed at 18 d, and the results showed that there was no difference in the survival rates of the five strains in the blank control. Under 125 mM NaCl treatment, the survival rates of OE17 and OE18 were significantly lower than that of WT, while the survival rate of atrgp1 was significantly higher than that of WT ( P < 0.05). Under 150 mM NaCl treatment, the survival rate of OE17 was significantly lower than that of WT, while the survival rate of the Arabidopsis mutant atrgp1 was significantly higher than that of WT ( P < 0.05) (Figure 13).

These results indicate that overexpression of MsRGP1 reduces the salt tolerance of Arabidopsis under salt stress, and are consistent with the results that OE17 is expressed at a higher level than OE18, while the salt tolerance of Arabidopsis mutants atrgp1 and atrgp2 is higher than that of the wild type.

(2) Effects of drought stress on Arabidopsis seed germination

MsRGP1 transgenic Arabidopsis (OE17 and OE18), Arabidopsis mutants ( atrgp1 and atrgp2 ) and wild-type Arabidopsis were plated on 1/2MS medium containing different concentrations of mannitol (300 mM and 400 mM) and observed. The germination rates of the five Arabidopsis lines were consistent under 0 mM mannitol treatment. Under 300 mM and 400 mM mannitol treatments, the germination rates of OE17 and OE18 were significantly lower than those of WT ( P < 0.05), while the germination rate of the Arabidopsis mutant atrgp1 was significantly higher than that of WT ( P < 0.05) (Figure 14). At the same time, biological statistics of the survival rates of the five strains were performed on the 18th day. The results showed that there was no difference in the survival rates of the five strains under the treatment of 0 mM mannitol, but the survival rates of OE17 and OE18 were significantly lower than those of WT under the treatment of 300 mM and 400 mM mannitol, the survival rate of atrgp1 was significantly higher than that of WT, and the survival rate of atrgp2 was significantly higher than that of WT under the treatment of 400 mM mannitol ( P < 0.05) (Figure 15).

These results suggest that MsRGP1 responds to osmotic stress and reduces the germination rate of Arabidopsis under drought stress. Therefore, overexpression of MsRGP1 reduces the tolerance of Arabidopsis to drought, while the Arabidopsis mutants atrgp1 and atrgp2 have higher tolerance to drought than wild-type Arabidopsis.

(3) Effects of alkaline stress on Arabidopsis seed germination

MsRGP1 transgenic Arabidopsis thaliana (OE17 and OE18), Arabidopsis mutants ( atrgp1 and atrgp2 ) and wild-type Arabidopsis thaliana were cultured in 1/2MS medium containing 5 mM NaHCO _3. The germination rates of the five lines were consistent under 0 mM NaHCO ₃ treatment. Under 5 mM NaHCO ₃ treatment, the germination rates of OE17 and OE18 were significantly lower than those of WT on the 2nd to 3rd day after culture, while the germination rate of atrgp1 was significantly higher than that of WT ( P < 0.05).

These results indicate that overexpression of MsRGP1 reduces the tolerance of plants to alkaline stress, and the Arabidopsis mutant atrgp1 has a higher tolerance to alkaline stress than the wild type.

(4) Effects of ABA stress on Arabidopsis seed germination

The results showed that there was no difference between the wild type, overexpression lines and mutants under 0 μM ABA treatment. Under 1 μM ABA treatment, the germination rates of OE17 and OE18 were significantly higher than that of WT, while the germination rates of the two mutant lines were significantly lower than that of the wild type ( P < 0.05). At the same time, biological statistics were performed on the green leaf rates of overexpression Arabidopsis and WT at 10 days of treatment, and Arabidopsis mutants and WT at 20 days of treatment. Under the blank control, there was no difference in the green leaf rate between overexpression Arabidopsis and WT, and between Arabidopsis mutants and WT. Under ABA stress, the green leaf rates of OE17 and OE18 were significantly higher than that of WT, while the greening rates of the two mutant lines were significantly lower than that of the wild type ( P < 0.05) (Figure 16).

These results indicate that MsRGP1 responds to ABA stress and may reduce the tolerance of Arabidopsis to salt and drought stress through the ABA signaling pathway.

4.2.2 Effects of saline-alkali, drought and ABA stress on Arabidopsis thaliana seedlings

The following results are obtained from the experimental results in Figure 17:

(1) Under 125 mM NaCl treatment, plant death was observed in both OE17 and OE18 lines, but this phenomenon did not exist in WT and mutant lines. In addition, the root length of the two overexpression lines was lower than that of the WT and mutant lines, while the root length of the two mutant lines was significantly higher than that of the WT ( P < 0.05). The fresh weight of the two mutant lines was significantly higher than that of the WT ( P < 0.05). This indicates that MsRGP1 responded to salt stress and showed a negative regulation phenomenon, which reduced the salt tolerance of Arabidopsis seedlings.

(2) Under 5 mM NaHCO ₃ alkaline stress, the leaves of Arabidopsis seedlings under alkaline stress showed that the degree of leaf chlorosis of OE17 and OE18 was higher than that of WT and mutant strains. There was no significant difference in root length and fresh weight among WT, overexpression Arabidopsis and mutant ( P < 0.05). This indicates that the response of MsRGP1 to alkaline stress is not obvious in Arabidopsis seedlings.

(3) The results under drought stress were consistent with those under salt stress. Under 300 mM mannitol treatment, the root length of the two mutant lines was significantly longer than that of the WT, while the root length of the MsRGP1 transgenic Arabidopsis was shorter than that of the WT ( P < 0.05). This indicates that overexpression of MsRGP1 reduces the drought tolerance of the plant, and the tolerance of the Arabidopsis mutant to drought stress is higher than that of the wild type.

(4) Under 30 μM ABA treatment, the roots of the mutant lines were shorter. Biostatistical analysis showed that the root lengths of OE17 and OE18 were significantly longer than those of WT, while the root lengths of the two mutant lines were significantly shorter than those of WT ( P < 0.05). This indicates that MsRGP1 responds to ABA stress and improves the tolerance of plants to ABA stress.

The above results indicate that overexpression of MsRGP1 reduces the tolerance of Arabidopsis thaliana to salinity and drought stress during the seedling stage, and enhances the tolerance to exogenous ABA stress. The Arabidopsis mutant has higher tolerance to salinity and drought stress than the wild-type Arabidopsis, and lower tolerance to exogenous ABA stress than the wild-type Arabidopsis.

4.2.3 Effects of drought stress on Arabidopsis seedling formation

Five Arabidopsis strains (WT, OE17, OE18, rgp1 , and rgp2 ) were subjected to drought treatment. After 12 days of drought treatment, the leaves of WT, OE17, and OE18 showed signs of chlorosis, wrinkles, and yellowing. Among them, OE17 showed the most severe chlorosis, while the leaves of atrgp1 and atrgp2 strains were still bright green and did not show signs of chlorosis. After 20 days of drought treatment, the plants were rehydrated and observed again 2 days after rehydration. It was found that compared with WT, OE17, and OE18, atrgp1 and atrgp2 strains had better growth and green leaves. However, no surviving plants were found in OE17 after rehydration, and the leaves of the plants showed signs of yellowing as a whole (Figure 18).

The results of biological statistics showed that after rehydration, the survival rate of WT was about 88.9%, while the survival rate of OE17 was as low as 14.8%, and the survival rate of OE18 was about 63%. The survival rates of the two overexpression lines were significantly lower than that of WT ( P < 0.05), and the survival rates of atrgp1 and atrgp2 lines were about 96.3%. This indicates that MsRGP1 reduces the tolerance of plants to drought, which is consistent with the previous experimental results of Arabidopsis seed germination and Arabidopsis seedling development under drought stress (Figure 19).

Example 5 Preparation of transgenic alfalfa with overexpression of MsRGP1 gene and analysis of stress resistance

5.1 Experimental Methods

5.1.1 Genetic transformation process of alfalfa and acquisition of positive plants

(1) Add 100 μL of pFGC-MsRGP1-eYFP Agrobacterium to 10 mL of liquid LB medium containing Kan (50 mg/L) and Rif (50 mg/L), and place it in a 28°C shaker at 200 rpm for 2-3 days until the bacterial solution becomes turbid and orange-yellow; add 100 mL of liquid LB containing Kan (50 mg/L) and Rif (50 mg/L) to a 250 mL sterile conical flask, then add 1-2 mL of the activated bacterial solution, and place it in a 28°C shaker at 200 rpm until the OD600 of the bacterial solution is 0.6-0.8.

(2) Add 100 mL of bacterial solution to two sterilized 50 mL centrifuge tubes, centrifuge at 6500 × g for 15 min at 4°C, discard the supernatant, add SH3a liquid medium and resuspend the suspension to an OD600 of 0.2-0.3, and transfer to a sterilized tissue culture bottle.

(3) Take 10 to 15 mature compound leaves of alfalfa. Wash the leaves under sterile conditions, use dd H ₂ O to clean the leaves, then use 75% alcohol to clean the leaves for about 1 min, and then use dd H ₂ O to clean the leaves 1 to 3 times. Soak the leaves in a 15 to 20% sodium hypochlorite solution with a drop of Tween 20, put it in a shaker at 50 rpm for 6 to 10 minutes, and finally use dd H ₂ O to clean the leaves 5 to 8 times.

(4) Transfer the washed leaves to the resuspension solution and evacuate for 5-10 min. Then place them in an ultrasonic device containing an ice-water mixture for about 5-15 sec. The specific time depends on the condition of the leaves. Vacuum again for 5-10 min. Transfer the leaves to sterilized filter paper to absorb excess water, and then transfer them to the SH3a co-culture solid medium. Then place them in a 24°C incubator and culture them in the dark for 22-30 h.

(5) Transfer the co-cultivated leaves to SH3a selective medium and continue culturing in a dark incubator at 24°C. Replace the medium every 12 to 16 days until expanded callus tissue is formed in 2 to 3 months.

(6) The expanded callus was transferred to MSBK medium and cultured at 24°C with 16 h light/8 h dark. The medium was replaced every 14 to 20 days for about 30 days.

(7) After the callus tissue produces green buds, it is transferred to SH9a medium. The medium is replaced every 20 to 30 days. After about 60 days, it is placed in SH9a medium without PPT and cultured until regenerated plants grow.

(8) Move the regenerated plants to the soil for cultivation. During the initial cultivation, ensure humidity and low light.

(9) Samples were taken from the leaves of regenerated alfalfa plants, and DNA was extracted and identified using specific primers 35S-F2 and MsRGP1-R1 (Table 4). Plants with the correct band size were positive.

(10) Leaves of positive plants were sampled, RNA of positive plants was extracted and reverse transcribed, and the reverse transcription products were used to verify the expression level by qRT-PCR test. Ms-actin-F and Ms-actin-R were internal reference gene primers, and Q-MsRGP1-F and Q-MsRGP1-R were primers for detecting the expression level of the target gene. The detailed procedure of qRT-PCR was carried out according to the instructions. Three replicates were set for each sample, and the data were analyzed using the 2-∆∆CT method. The results are presented in the form of mean ± standard deviation (SD). The qRT-PCR primer information is listed in the table (Table 4). The 2 to 4 strains with the highest expression levels were selected for cultivation and cut at the same time as the wild type for subsequent experiments.

5.1.2 Functional analysis of MsRGP1 transgenic alfalfa under salt and drought stress

About 1 to 2 months after the wild-type alfalfa and transgenic alfalfa were cut, the alfalfa with the same growth after cutting was selected and transferred to new soil (vermiculite) for salt stress and drought stress treatment.

(1) Salt stress treatment method: WT, OE26 and OE27 were subjected to salt treatment (300 mM NaCl in Hoagland nutrient solution), with four replicates for each strain. The phenotypes were observed and photographed from the first day of treatment.

(2) Drought treatment method: WT, OE26 and OE27 were used for drought treatment (natural drought), with four replicates for each strain. The phenotypes were observed and photographed from the first day of treatment.

Table 4 Primer information

5.2 Experimental Results and Analysis

5.2.1 Obtaining MsRGP1 transgenic alfalfa

Through the method of alfalfa genetic transformation, 42 regenerated plants were finally obtained. After PCR identification, there were 23 positive plants, with a positive rate of about 54.76%. Subsequently, the expression of positive plants was verified by qRT-PCR technology (Figure 20), and OE26 and OE27 were selected for subsequent experiments and cuttage was performed on them.

5.2.2 Functional analysis of MsRGP1 transgenic alfalfa under salt and drought stress

Two MsRGP1- overexpressing alfalfa lines (OE26 and OE27) and wild-type alfalfa (WT) were selected for cuttings. About 45 days after cuttings, MsRGP1 transgenic alfalfa and wild-type alfalfa with consistent growth were selected for salt treatment and drought treatment.

Before salt stress treatment, OE26 and OE27 had no phenotypic differences from WT. After 10 days of treatment with 300 mM NaCl aqueous solution, the growth and development of OE27 was restricted during the salt treatment period, and lodging and leaf wilting occurred. A few leaves of OE26 showed wrinkles, while WT did not show obvious changes. At 15 days of salt treatment, the stems and leaves of OE27 lost water and wilted and fell off. The leaves of OE26 began to wilt on a large scale, while WT still showed no obvious changes. At 20 days of salt treatment, OE27 was completely inactivated, OE26 showed lodging and its leaves and stems were basically wilted, while only a few leaves of WT showed wilting (Figure 21). These results indicate that MsRGP1 reduces the tolerance of alfalfa to salt stress.

About 45 days after cutting, MsRGP1 transgenic alfalfa (OE26 and OE27) and WT with consistent growth were selected for drought treatment. Before drought treatment, there was no phenotypic difference among WT, OE26 and OE27. After 15 days of drought treatment, WT did not change significantly, while the leaves of OE26 and OE27 showed signs of water loss and wilting, among which the leaves of OE27 wilted more severely. At 17 days of drought treatment, the leaves of OE27 basically lost water and wilted and fell off. The wrinkles of OE26 leaves became more severe but did not wilt, while only a small number of WT leaves wilted. At 20 days of drought treatment, the leaves of OE26 and OE27 had completely lost water and shrank, while the leaves of WT wilted and lost water, and were relatively less affected by drought stress (Figure 22). The results showed that MsRGP1 reduced the tolerance of alfalfa to drought stress.

It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims (3)

1. An application of MsRGP1 protein in improving drought resistance and/or salt tolerance of plants, wherein the plant is alfalfa or Arabidopsis thaliana, characterized in that the amino acid sequence of the MsRGP1 protein is as shown in SEQ ID NO: 1, and the drought resistance and salt tolerance of the plant are improved by downregulating the expression level of the MsRGP1 gene in the plant or knocking out the MsRGP1 gene, and the nucleotide sequence of the MsRGP1 gene is as shown in SEQ ID NO: 2. 2. Application of the MsRGP1 gene in improving drought resistance and/or salt tolerance of plants, wherein the plant is alfalfa or Arabidopsis thaliana, and wherein the nucleotide sequence of the MsRGP1 gene is as shown in SEQ ID NO: 2, and the drought resistance and salt tolerance of the plant are improved by down-regulating the expression level of the MsRGP1 gene in the plant or knocking out the MsRGP1 gene. 3. A method for preparing a transgenic plant, characterized in that the method is the following (1) or (2): (1) By reducing the expression of the MsRGP1 gene in wild-type plants, plants with stronger salt stress tolerance and drought resistance than wild-type plants were obtained; (2) By promoting the expression of the MsRGP1 gene in wild-type plants, plants with lower salt stress tolerance and drought resistance than wild-type plants were obtained. The plant is alfalfa or Arabidopsis thaliana, and the nucleotide sequence of the MsRGP1 gene is shown in SEQ ID NO:2.