Application of MsGADP2 gene in improving plant salt stress resistance

Abstract

This invention discloses the application of the MsGADP2 gene in improving plant salt stress resistance, belonging to the field of biotechnology. The invention experimentally verifies the correlation between the MsGADP2 gene and plant salt tolerance, demonstrating that regulating MsGADP2 gene expression can effectively regulate plant salt stress resistance. The method provided by this invention has significant reference value for cultivating and obtaining salt-tolerant plants.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to the application of the MsGADP2 gene in improving plant salt stress resistance. Background Technology

[0002] Alfalfa (Medicago sativa L.) is a perennial herbaceous plant belonging to the genus Medicago in the legume family. It is now widely introduced and cultivated and is known as the "King of Forage".

[0003] Drought and salt stress are major limiting factors for crop production. Studies have shown that global desertification and salinization are increasingly severe, affecting more than 10% of arable land and causing an average yield reduction of more than 50% for major crops. Therefore, elucidating the tolerance mechanisms of plants to drought stress and high salinity is an important issue in agricultural production. Alfalfa is mainly grown in arid and semi-arid regions of the north. Although it adapts well to various climatic and soil conditions, including arid and saline environments, drought and high salinity still limit the increase of alfalfa yield.

[0004] Poly(ADP-ribose) polymerase (PARP) was first described in plants 15 years ago. Its mediated protein poly(ADP-ribosylation) participates in various cellular processes, including cell survival and death, transcription, metabolism, energy homeostasis, DNA repair, DNA replication, RNA homeostasis, telomere integrity, and cell division. Increasing evidence suggests that PARP participates in multiple physiological processes in plants, including growth, development, and stress responses. Studies have demonstrated that Arabidopsis thaliana PARP1 and PARP2 participate in Arabidopsis growth and development, as well as responses to biotic stress, by regulating DNA repair and transcription. Although there are reports of PARP participating in plant responses to abiotic stress, research on PARP responses to salt stress is limited, and no reports have been made in alfalfa.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the MsGADP2 gene in improving plant salt stress resistance.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides an alfalfa MsGADP2 protein containing the amino acid sequence shown in (a) or (b) below:

[0009] (a) The amino acid sequence shown in SEQ ID NO.2;

[0010] (b) Proteins derived from (a) whose amino acid sequence shown in SEQ ID NO.2 has been modified by substitution and / or deletion and / or addition of one or more amino acid residues and are associated with plant salt stress resistance.

[0011] Secondly, the present invention provides a nucleic acid molecule that encodes the aforementioned alfalfa MsGADP2 protein.

[0012] In some embodiments, the nucleotide sequence of the above-mentioned nucleic acid molecule is shown in SEQ ID NO.1.

[0013] Thirdly, the present invention provides a recombinant vector containing the aforementioned nucleic acid molecules.

[0014] Fourthly, the present invention provides a recombinant bacterium containing the above-mentioned recombinant vector.

[0015] Fifthly, this invention provides the application of alfalfa MsGADP2 protein and its encoding gene in improving the plant's salt stress resistance.

[0016] In some embodiments, the above-mentioned plants include Arabidopsis thaliana and alfalfa.

[0017] In some embodiments, the above applications include regulating lateral root development in plants under salt stress.

[0018] In a sixth aspect, the present invention provides a method for improving the salt stress resistance of plants, comprising introducing the above-mentioned nucleotide molecules into the genome of a target plant to obtain a transgenic plant; the above-mentioned plants include Arabidopsis thaliana and alfalfa.

[0019] In a seventh aspect, the present invention provides a plant breeding method comprising: increasing the content and / or activity of the aforementioned alfalfa MsGADP2 protein in plants, thereby enhancing the plants' resistance to salt stress; wherein the aforementioned plants include Arabidopsis thaliana and alfalfa.

[0020] The present invention has the following beneficial effects:

[0021] This invention constructs an overexpression fusion vector, transiently transforms tobacco epidermal cells, and performs subcellular analysis. The results show that MsGADP2 is located in the cell nucleus. Then, using the flower-dip transformation technique, MsGADP2 is transformed into Arabidopsis thaliana, verifying its salt tolerance. Germination rate and root length experiments under salt stress conditions confirm that the salt tolerance of MsGADP2 transgenic Arabidopsis thaliana on salt plates is significantly better than that of the wild type. Furthermore, transient transformation using alfalfa hairy roots verifies that the MsGADP2 gene plays a positive regulatory role in the response to salt stress and also confirms the correlation between MsGADP2 overexpression and the induction of an antioxidant system to protect cells from oxidative damage. Therefore, it can be shown that the MsGADP2 gene can enhance the plant's tolerance to salt stress. This method of regulating plant salt stress resistance by controlling MsGADP2 gene expression has important reference and learning value for cultivating and obtaining salt-tolerant plants. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This refers to the cloning of the MsGADP2 gene and the construction of the pYES2 yeast expression vector in Example 1;

[0024] Figure 2 This refers to the growth phenotype of MsGADP2-transformed yeast cells under drought and salt stress in Example 2.

[0025] Figure 3 This refers to the cloning of the MsGADP2 gene and the construction of the pBI12::MsGADP2::EGFP overexpression vector in Example 3;

[0026] Figure 4 Figure 3 shows the construction of the RNAi-MsGADP2 recombinant vector. Figure A shows the identification of the MsGADP2 attB-PCR product; Figure B shows the LR reaction monoclonal PCR detection. The primers are RNAi-F / RNAi-R and GUSlinker-F / GUSlinker-R, respectively.

[0027] Figure 5This image shows the subcellular localization of the MsGADP2 protein in Example 4. EGFP: a protein with green fluorescence; PM: a plasma membrane localization marker protein; DAPI: a nuclear dye; Bright field; Merge: an overlay of GFP, PM, and Bright field.

[0028] Figure 6 For the identification of the MsGADP2 positive Arabidopsis strain in Example 5, A: Detection results using NPT-F / NPT-R resistance primers; B: Detection results using 35S-F / MsGADP2-R1 chimeric primers; "M" represents Marker; "-" represents blank control; "+" represents pBI121::MsGADP2::EGFP vector plasmid; "Col-0" represents wild type;

[0029] Figure 7 To identify the expression level of MsGADP2 transgenic Arabidopsis thaliana in Example 5;

[0030] Figure 8 For the germination rate statistics of MsGADP2 transgenic Arabidopsis thaliana under salt stress in Example 5, A: germination rate phenotype of MsGADP2 transgenic Arabidopsis thaliana and Col-0 wild-type Arabidopsis thaliana on control plates; B: germination rate phenotype of MsGADP2 transgenic Arabidopsis thaliana and Col-0 wild-type Arabidopsis thaliana on 150mM NaCl plates; C: germination rate statistics of the corresponding genotype germination phenotype in A; D: germination rate statistics of the corresponding genotype germination phenotype in B. The error is the standard error of three biologically repeated occurrences.

[0031] Figure 9 The table shows the root length phenotypes of MsGADP2 transgenic Arabidopsis thaliana under salt stress in Example 5. A: Growth phenotypes of MsGADP2 transgenic Arabidopsis thaliana and Col-0 wild-type Arabidopsis thaliana on control plates; B: Growth phenotypes of MsGADP2 transgenic Arabidopsis thaliana and Col-0 wild-type Arabidopsis thaliana on 150 mM NaCl plates; C: Relative lateral root length statistics for the corresponding transgenic lines in A; D: Lateral root number statistics for the corresponding transgenic lines in B; The error is the standard error of three biologically repeatable occurrences, **P < 0.01;

[0032] Figure 10 In Example 6, PCR detection of MsGADP2 transgenic alfalfa hairy roots was performed. A: RNAi transgenic hairy root detection results; B: Overexpression of MsGADP2 transgenic hairy root detection results; In the figure, "M" represents Marker; "-" represents blank control; "+" represents pBI121-MsGADP2:EGFP vector plasmid.

[0033] Figure 11The image shows the root length phenotype of MsGADP2 transgenic alfalfa hairy roots on a plate under salt stress in Example 6. EGFP: a protein with green fluorescence; PM: a plasma membrane localization marker protein; DAPI: a nuclear dye; Bright field; Merge: an overlay of GFP, PM, and Bright field; A: growth phenotypes of EV, MsGADP2, and RNAi transgenic alfalfa on control and salt stress plates; B: lateral root length statistics for the corresponding genotype plants in A; C: lateral root number statistics for the corresponding genotype plants in A; errors are the standard errors of three biologically repeated occurrences; **P<0.01;

[0034] Figure 12 To identify the expression level of the MsGADP2 gene in hairy roots transformed under salt stress in Example 6;

[0035] Figure 13 Figure 6 shows the detection of reactive oxygen species (ROS) in EV and transgenic hairy roots under control and salt treatment conditions; Figure A shows ROS images; Figure B shows fluorescence intensity.

[0036] Figure 14 This document evaluates the growth phenotype of yeast cells under H2O2 treatment and the root length phenotype of MsGADP2 transgenic alfalfa hairy roots under salt treatment and exogenous H2O2 and GSH stress in Example 6. Figure A shows the yeast growth phenotype under H2O2 treatment; Figure B shows the root length phenotype of MsGADP2 transgenic alfalfa hairy roots under salt treatment and exogenous H2O2 and GSH stress; Figures C and D show the lateral root number and lateral root length statistics for the corresponding lines in Figure B. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0038] In this invention, the inventors cloned the MsGADP2 gene for the first time from alfalfa (Medicago sativa L.) of the genus Medicago in the legume family. Experiments have shown that the MsGADP2 protein is related to the stress resistance of plants, especially the salt stress resistance of alfalfa. This gene and its encoded protein can be used to improve the tolerance of plants to salt stress, providing new ideas and methods for studying salt tolerance-related regulatory genes.

[0039] The amino acid sequence of MsGADP2 protein is shown in SEQ ID NO.2, but is not limited to the amino acid sequence shown in SEQ ID NO.2. It can also be a protein derived from SEQ ID NO.2 with one or more amino acid residues substituted and / or deleted and / or added, and associated with plant salt stress resistance.

[0040] In addition, to facilitate the purification or detection of the above proteins, a tag protein can be attached to the amino or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO.2.

[0041] The nucleotide sequence of the MsGADP2 gene in this invention is shown in SEQ ID NO.1.

[0042] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein MsGADP2 of this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that possess 90% or more of the nucleotide sequence identity with the protein MsGADP2 isolated in this invention, provided they encode and function as protein MsGADP2, are derived from and equivalent to the nucleotide sequence of this invention.

[0043] The present invention also provides a recombinant vector containing the above-mentioned nucleic acid molecules.

[0044] Specifically, the recombinant vectors mentioned above include any of the following vectors: target gene cloning vector (for preserving and cloning target genes, such as E. coli plasmids), intermediate cloning vector (constructed by inserting T-DNA fragments into E. coli plasmids and target genes, marker genes, etc., and is the basic plasmid for constructing intermediate expression vectors), intermediate expression vector (an intermediate vector containing plant-specific promoters, used as a plasmid for constructing transformation vectors), and / or plant gene transformation vector (a vector used for introducing target genes into plant cells).

[0045] The present invention also provides a recombinant bacterium containing the above-mentioned recombinant vector.

[0046] The present invention also provides a method for preparing the above-mentioned MsGADP2 protein, comprising: encoding the above-mentioned nucleic acid and preparing it through biological expression and / or artificial synthesis.

[0047] The present invention also provides the application of the above-mentioned salt stress resistant protein and its encoding gene in improving the salt stress resistance of plants.

[0048] Specifically, methods to improve the salt stress resistance of plants include: introducing the above-mentioned nucleic acid or the CDS region of the nucleic acid having the base sequence shown in SEQ ID NO.1 into the genome of the target plant to obtain transgenic plants; and artificially cultivating the transgenic plants to allow them to grow naturally.

[0049] Furthermore, the aforementioned nucleic acid or CDS regions are introduced into plants using a recombinant expression vector.

[0050] The aforementioned plants include Arabidopsis thaliana and alfalfa.

[0051] The protein and its encoding gene provided by this invention can genetically optimize plant varieties, enhance their tolerance to salt stress, and promote plant growth under salt stress conditions, especially the normal growth of lateral roots. Therefore, constructing plants expressing the MsGADP2 gene through transgenic technology can be applied to salt-tolerant plant breeding and has good application prospects.

[0052] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0053] The experiments of this invention used seeds of Nicotiana benthamiana, alfalfa 'Zhongmu No. 1', and Arabidopsis thaliana (Col-0), all of which were stored in the National Key Laboratory of Grass Seed Innovation and Grassland Agro-ecosystems at Lanzhou University.

[0054] The Agrobacterium GV3101 and Escherichia coli DH5α competent cells used were purchased from Beijing TransGen Biotech Co., Ltd. The Agrobacterium rhizogenes Ar.Qual strain, expression vector plasmid pBI121::EGFP vector and RNAi vector pANDA35HK were all preserved by the National Key Laboratory of Grass Seed Innovation and Grassland Agro-ecosystem at Lanzhou University. The Saccharomyces cerevisiae INVSC1 strain was purchased from Yikeshu Company.

[0055] Example 1

[0056] This example demonstrates gene cloning and expression vector construction, as detailed below:

[0057] (1) Primer design and candidate gene cloning

[0058] CDS sequence data of candidate genes (SEQ ID NO. 1) were extracted from the alfalfa genome database, and gene primers were designed using Snapgene software. Primers were designed with BamHI and KpnI restriction sites for ligation of the pYES2 vector, and with SalI and SpeI restriction sites for ligation of the pBI121::EGFP vector. A double digestion method was used to design forward and reverse primers. Primer sequences are shown in Table 1. All primers were synthesized by Xi'an Qingke Biotechnology Co., Ltd. MCLAB 1-5 was used... TM The reaction system was amplified using 2×High-Fidelity Master Mix (Qingke, Xi'an). Table 2 shows the reaction system.

[0059] Table 1 Gene Primer Information

[0060]

[0061]

[0062] Table 2 Gene Cloning PCR Reaction System

[0063]

[0064] Table 3 PCR reaction procedure

[0065]

[0066] The PCR reaction procedure is shown in Table 3. A 1.5% agarose gel was prepared, and the electrophoresis detection current was 400 mA, the voltage was 140 V, and the electrophoresis time was 30 min. After scanning with a WD-9413B gel imaging analysis system (Liuyi, Beijing), the target DNA fragment was excised from the gel and recovered using a DNA gel recovery kit (TransGen, Beijing). The fragment was ligated with the pEASY-Blunt SimpleCloning Kit vector at 37℃ for 15 min, and the ligation product was transformed into *E. coli* DH5α. PCR detection of the bacterial culture was performed using the M13-F / M13-R universal primers, followed by sequencing confirmation.

[0067] (2) Construction of yeast expression vector

[0068] The pYES2 yeast expression vector and the target fragment were digested with enzymes according to the enzyme digestion systems in Tables 4 and 5, respectively. After vortexing and mixing, the reaction mixtures were incubated in a water bath for 3 hours, followed by gel electrophoresis for detection. The target band was then excised and recovered from the gel. Using T4 DNA ligase (M0202, New England Biolabs Beijing Co., Ltd.), the recovered target fragment was ligated to the pYES2 yeast expression vector fragment according to the system in Tables 5-6. The ligation was then performed on *E. coli*, and after overnight culture to produce single colonies, PCR detection was performed. The correctly sequenced single colonies were then stored and plasmids were extracted for later use.

[0069] Table 4. Double enzyme digestion system for expression vector

[0070]

[0071]

[0072] Table 5. Double enzyme digestion reaction system for the target fragment

[0073]

[0074] Table 6 Connection Reaction System

[0075]

[0076] Based on the empty vector map and gene sequence, two restriction sites, BamHI and KpnI, were selected to amplify the CDS sequence of the MsGADP2 gene. Figure 1 A) The pYES2 vector and the target gene fragment were ligated using double enzyme digestion to construct an expression vector. After transformation into *E. coli*, colony PCR was performed for identification. The primers for colony PCR identification were T7-F / MsGADP2-R1 (chimeric primers, not full-length CDS sequences). Correctly identified positive clones were sent for sequencing after shaking. Plasmids were extracted from and preserved from the successfully sequenced clones. Figure 1 B).

[0077] Example 2

[0078] This example demonstrates the heterologous expression and salt tolerance function of yeast, as detailed below:

[0079] (1) Transform the constructed vector plasmid and the pYES2 empty vector plasmid into competent cells of INVSC1 yeast.

[0080] (2) To identify the correct pYES2 empty vector and positive yeast strain containing the target gene, single clones were cultured in 10 mL SC-Ura glucose liquid medium and incubated on a shaker at 28°C and 200 rpm for 24 h.

[0081] (3) Determination of bacterial culture OD 600The bacterial culture was then diluted to 0.4 with 10 mL of SC-Ura galactose-induced expression medium and cultured at 28°C and 200 rpm for 36 h to induce exogenous gene expression.

[0082] (4) Adjust the bacterial culture to OD 600 =1.0, take 500 μL, centrifuge briefly at 12000 rpm, and then discard the supernatant.

[0083] (5) After centrifugation, the yeast cells were resuspended in equal volumes of 4M NaCl, 4M sorbitol, 15mM H2O2 and sterile water (control), and cultured in a shaker at 28℃ and 200rpm for 72h, 120h, 120h and 96h respectively. The culture time of the control was the same as that of the treatment.

[0084] (6) Dilute the above-treated bacterial solutions according to dilution gradients of 1, 10, and 10. -1 10 -2 10 -3 10 -4 10 -5 These six gradients were diluted with sterile water in 2 mL tubes, and 5 μL of bacterial suspension was taken and spotted onto SC-Ura glucose solid medium. The experiment was conducted with three technical replicates and three biological replicates. The tubes were sealed and incubated upside down at 28°C for 3-4 days. During the incubation process, real-time photographic records were taken of the colony growth.

[0085] The results showed that in the control group, there was no significant difference in growth between transgenic yeast cells and empty yeast cells. However, after stress with 4M NaCl and 4M sorbitol, the transgenic yeast cells showed significantly better growth than the empty yeast cells. Figure 2 This indicates that MsGADP2 enhances the tolerance of yeast strains to drought and salt stress through its transgenic expression, and preliminarily demonstrates that the MsGADP2 salt-drought tolerant candidate gene plays a positive regulatory role in yeast cells' response to drought and salt stress.

[0086] Example 3

[0087] This example demonstrates the cloning and overexpression vector construction of MsGADP2, as detailed below:

[0088] The overexpression vector pBI121::EGFP, preserved in our laboratory, was selected. Based on the vector map and gene sequence, Sal I and Spe I restriction enzyme sites were chosen, and the vector was constructed using a double digestion method. Primers were designed using Snapgene software (Table 1) and synthesized by Xi'an Qingke Biotechnology Co., Ltd. MCLAB 1-5 was used... TMThe target fragment was amplified using 2× High-FidelityMaster Mix amplification enzyme (Qingke, Xi'an), following the amplification system described in Example 1. DNA products from the gel were recovered using a gel extraction kit (TransGen, Beijing). The pEASY-Blunt Simple Cloning Kit vector was ligated, and the ligation product was transformed into *E. coli* DH5α. Single clones were picked and tested using M13-F and MsGADP2-R1 (non-full-length primer sequences) before being sent to the company for sequencing confirmation. After successful sequencing, the plasmid was extracted and subjected to double enzyme digestion with the pBI121::EGFP expression vector plasmid. The reaction system was as described in Example 1. After the enzyme digestion was completed, the fragment was confirmed by electrophoresis. The digested target fragment was ligated with the digested fragment of the pBI121::EGFP expression vector using T4 DNA ligase (M0202, New England Biolabs Beijing Co., Ltd.). Subsequently, the fragment was transformed into competent E. coli cells and cultured overnight at 37°C. After colony PCR detection, the colony was sent for sequencing after successful detection. The single clone with correct sequencing was stored and the plasmid was extracted for later use.

[0089] (1) Obtaining the target fragment

[0090] Gateway cloning technology was used to design primers, and the constructed overexpression vector plasmid was used as a template to utilize MCLAB1-5. TM PCR amplification was performed using 2×High-Fidelity Master Mix enzyme. The PCR products were then recovered using a gel extraction kit and set aside for later use.

[0091] Table 7 Primer Information

[0092]

[0093] (2) Target fragment connection entry carrier

[0094] The connection reaction system is as follows:

[0095]

[0096] The ligation product was transformed into Escherichia coli DH5α, plated, and incubated upside down in a 37°C incubator for 16 h. Positive single clones were detected using universal primers M13F and M13R and sent for sequencing. Single clones with correct sequencing were cultured, and plasmids were extracted and stored at -20°C for later use.

[0097] The fusion expression vector pBI121-MsGADP2:EGFP was successfully constructed. The gene cloning primers were MsGADP2-F / MsGADP2-R, and the colony PCR detection primers were the chimeric primers 35S-F / MsGADP2-R1 (full-length primers without the CDS sequence). Figure 3 As shown.

[0098] (3) Constructing RNAi recombinant vectors via LR reaction

[0099] First, the target vector pANDA35HK was transformed into E. coli DH5α for propagation. After verification, plasmid extraction was performed. The concentrations of the target vector and the entry vector were determined to be 100 ng / μL. Then, an LR reaction was performed, with the following system:

[0100] 2×Gateway LR Clone Enzyme mix 5μL

[0101] Entry Clone (100ng / μL) 3μL

[0102] Target vector (0.5 μg / μL) 2 μL

[0103] After thoroughly mixing the above reaction system, centrifuge rapidly and incubate overnight at 25°C. Add 1 μL of 10× Proteinase K Solution, vortex to mix, and incubate at 37°C for 20 min. Then transform it into Escherichia coli DH5α. After transformation, take 200 μL of culture and spread it on LB solid medium containing kanamycin (Kan) antibiotic at a final concentration of 50 mg / L. Invert the medium and incubate at 37°C for 16 hours. Extract plasmids from successfully colonized single clones and store them at -20°C for later use.

[0104] RNAi vectors were constructed using the Gateway method. The attB-PCR product of MsGADP2 was obtained via PCR and ligated into the entry vector. After positive clone identification, the sequenced entry clone was subjected to an LR reaction with the target expression vector pANDA35HK. Single-clone detection was then performed. Figure 4 Plasmid extraction from positive bacterial cultures yields the RNAi recombinant vector for the MsGADP2 gene (RNAi-MsGADP2).

[0105] (4) Transformation and identification of Agrobacterium

[0106] 1) Preparation of Agrobacterium tumefaciens Ar.Qual competent cells

[0107] 2) Electrocution method conversion

[0108] 3) Thermal shock conversion

[0109] The specific steps of the above method are referenced from the literature (Identification of ERF transcription factor in Osmanthus fragrans and its functional analysis in response to drought stress, Wei Na, 2024).

[0110] Example 4

[0111] This embodiment describes subcellular localization in tobacco, and the specific steps are as follows:

[0112] (1) The Agrobacterium strain containing plasmids that was successfully detected in Example 3 was shaken in 10 mL of LB liquid medium (containing 50 mg / L Kan and 50 mg / L rifampin) and cultured overnight on a shaker at 28°C and 200 rpm.

[0113] (2) OD in bacterial culture was measured using an ultraviolet spectrophotometer. 600 The value should be between 0.5 and 1. After centrifuging at 2400g for 15 minutes, collect the bacterial cells and remove the supernatant.

[0114] (3) To increase the OD of the new bacterial culture 600 When the pH reached the range of 0.4-0.6, we resuspended the bacterial cells in a resuspension solution and prepared the culture using MS liquid medium. For each 100 mL volume, we added 1 mL of 1M MgCl₂·6H₂O (final concentration 10 mM), 1 mL of 1M 2-(N-morpholino)ethanesulfonic acid (MES) (final concentration 10 mM), and 10 μL of 1M acetylsyringone (AS) (final concentration 100 μM), and adjusted the pH to 5.8. After storing at room temperature for 2-3 hours, infection could begin.

[0115] (5) Before the tobacco seedlings enter the soaking stage, they should be sprayed with enough water and cultivated under appropriate light conditions to ensure that the stomata of the tobacco leaves can be fully opened.

[0116] (7) Using a syringe with the needle removed, draw 1 mL of the suspension and slowly inject it into the infected area on the underside of the leaf.

[0117] (8) After spraying water on the soaked tobacco leaves, cover them with a black plastic bag and darken them overnight before opening the plastic bag.

[0118] (9) Two to three days after injection, we collected leaf samples from the stained area using a perforator and prepared slides. We then took photographs using a confocal microscope after adjusting to the optimal field of view.

[0119] The results are as follows Figure 5 As shown, the pBI121::MsGADP2::EGFP fusion protein was observed to be localized on both the cell membrane and the nucleus. This indicates that the MsGADP2 protein may perform its function in both the cell membrane and the nucleus.

[0120] Example 5

[0121] This example demonstrates the salt tolerance function of transgenic Arabidopsis thaliana.

[0122] (1) Arabidopsis genetic transformation: transgenic Arabidopsis seedlings were obtained by the flower dipping method. The main steps are referred to (Identification of ERF transcription factor in Osmanthus fragrans and its function in response to drought stress, Wei Na, 2024).

[0123] (2) Identification and expression level analysis of transgenic Arabidopsis thaliana seedlings

[0124] DNA was extracted from 10 transgenic Arabidopsis thaliana lines of generation T1 using a plant genomic DNA extraction kit (Tiangen, Beijing). PCR was performed using chimeric primers 35S-F / MsGADP2-R1 to verify positive plants. After identification as positive, RNA was extracted using the RNAEasy Fast Plant Tissue RNA Rapid Extraction Kit (Tiangen, Beijing). Next, reverse transcription was performed using the FastKing cDNA First-Strand Synthesis Kit (Tiangen, Beijing) to obtain cDNA. The relative expression level of MsGADP2 in transgenic Arabidopsis thaliana was then determined using qRT-PCR, as shown in the tables below. The qRT-PCR reaction system and procedure are shown in Tables 8 and 9. The reaction was performed for 40 cycles with three technical replicates (Wu et al., 2018). -ΔΔCT The computational method processes the data to obtain the relative expression levels of genes.

[0125] Table 8 qRT-PCR reaction system

[0126]

[0127] Table 9 qRT-PCR reaction procedure

[0128]

[0129]

[0130] Positive Arabidopsis thaliana transgenic with the MsGADP2 gene were detected by PCR using genomic DNA extraction. The kanamycin resistance gene detection primers NPT-F / NPT-R and the promoter-target gene chimeric primer 35S-F / MsGADP2-R1 were used to detect positive plants, resulting in 10 overexpressing positive Arabidopsis thaliana lines. Figure 6 Further, RNA was extracted from these 10 transgenic Arabidopsis thaliana lines and reverse transcribed into cDNA. The expression level of MsGADP2 was then detected using qRT-PCR. The results showed that the expression level of MsGADP2 in all 10 transgenic lines was significantly higher than that in the Col-0 line, with the highest expression levels observed in the overexpression lines OE2, OE3, and OE10. Figure 7).

[0131] (3) Evaluation of salt tolerance in transgenic Arabidopsis thaliana

[0132] Seeds from the three representative Arabidopsis lines with the highest expression levels of the MsGADP2 gene (OE2, OE3, and OE10) and the wild-type Col-0 were sterilized and then evenly spread on 1 / 2 MS medium and 1 / 2 MS medium containing 150 mM NaCl using a 10 μL pipette tip. After sealing, the seeds were treated at 4°C for 3 days, then transferred to a light incubator at 22°C and 60% relative humidity for 7 days. Germination rate was recorded daily, with three biological replicates for each treatment. The results were averaged and the standard deviation was calculated. Then, three transgenic lines with consistent root length and the wild-type Col-0 line were selected and transferred to 1 / 2 MS medium and 1 / 2 MS medium containing 150 mmol / L NaCl, respectively. The Arabidopsis plates were placed vertically in a light incubator with a growth environment of 22°C, 16 hours of light, and 8 hours of darkness. Observe the root growth of Arabidopsis seedlings regularly. After about two weeks, take photos of the growth phenotype and measure the root length. Take the average value of the results and calculate the standard deviation.

[0133] The results are as follows Figure 8 As shown, on 1 / 2 MS plates, the germination rates of wild-type and transgenic lines were basically the same; however, on 150 mM NaCl plates, the germination rate of MsGADP2 transgenic Arabidopsis lines was significantly greater than that of wild-type. The results are as follows... Figure 9 As shown, on 1 / 2 MS plates, the root length and number of lateral roots of wild-type and transgenic lines were basically the same; however, on 150 mM NaCl plates, the relative lateral root length and number of lateral roots of transgenic lines were significantly greater than those of wild-type.

[0134] Example 6

[0135] This example verifies the salt tolerance of alfalfa hairy roots, as detailed below:

[0136] 1. Transient transformation of alfalfa hairy roots

[0137] The function of the MsGADP2 gene in alfalfa was validated using the established hairy root transformation system in the laboratory. The specific steps are as follows:

[0138] (1) Seed cleaning: Select plump alfalfa 'Zhongmu No. 1' seeds, add concentrated sulfuric acid and shake for 3 minutes, then remove the concentrated sulfuric acid. Wash the seeds 5 times with ddH2O in a clean bench, then treat with 6% sodium hypochlorite solution and shake for 5 minutes, then wash with ddH2O water until colorless. Spread the seeds on FA plates and incubate in a 25℃ light incubator for two weeks.

[0139] (2) Infection: About two weeks later, when the seedlings have grown their third cotyledon, gently grasp the alfalfa seedlings with sterilized tweezers and cut them at the junction of the rootstock with a scalpel. Gently grasp the seedlings with the cut roots with tweezers and dip them in a small amount of Agrobacterium rhizogenes transgenic EV, MsGADP2 and RNAi. Then spread them on water agar plates, placing about 12 seedlings on each plate. Seal the plates and place them vertically in a 22°C incubator until roots emerge.

[0140] (3) Salt stress: Infected plants were divided into two groups. The treatment groups were cultured on water agar medium supplemented with 100 mM NaCl; 100 mM NaCl + 10 mM ABA; 100 mM NaCl + 10 mM ABA inhibitor; 100 mM NaCl + 0.1 μM NAA; 100 mM NaCl + 10 mM H2O2; and 100 mM NaCl + 400 μM GSH. The control group was cultured on water agar medium. After two weeks, the lateral root phenotype was observed and photographed. Samples were then taken from both the treatment and control groups. The expression level of MsGADP2 in the transgenic hairy roots was measured, and the reaction system and procedure were the same as in Example 4.

[0141] The constructed pBI121::MsGADP2::EGFP expression vector and RNAi vector were first transformed into *Agrobacterium rhizogenes*, and then transiently expressed in alfalfa hairy roots using alfalfa hairy root transformation technology. The control was an empty vector pBI121::EGFP control. After infecting *Agrobacterium rhizogenes* with alfalfa, the roots were placed vertically in a light-controlled culture room on water agar medium. After two weeks of root emergence, a small amount of hairy roots infected with the pBI121::MsGADP2::EGFP expression vector and RNAi vector were cut for positive root identification. The results showed that the fragment amplified through the transgenic hairy root DNA was consistent in size with the target band. Figure 10 ).

[0142] To further investigate the function of MsGADP2 in lateral root development, transgenic lines that had developed roots were transferred to control and stress plates for culture. After one week, their phenotypes were observed and photographed. The results showed that, compared to the control treatment, the number of lateral roots in hairy roots overexpressing the transgenic line under salt stress was significantly greater than that in EV and RNAi (…). Figure 11 This indicates that overexpression of MsGADP2 plays a positive regulatory role in lateral root development. We measured the expression level of MsGADP2 in transgenic hairy roots under control and salt stress treatments. We found that the expression level in transgenic hairy roots before and after treatment was significantly higher than that in wild-type and RNAi-treated roots. Figure 12).

[0143] 2. Reactive oxygen species staining of transgenic alfalfa hairy roots

[0144] To detect ROS levels in the hairy roots of transgenic alfalfa, the dye OxyBURST green H2DCFDA was used. Hairy roots of untreated and control alfalfa seedlings treated with 100 mM NaCl for 5 hours were analyzed. First, lateral roots from salt-treated and control seedlings were collected separately in 1.5 mL centrifuge tubes. H2DCFDA was first dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mM, then diluted to a final concentration of 50 mM with sample buffer (10 mM Tris-HCl, 50 mM KCl, pH 7.2). The samples were then immersed in 0.01% Tween 20 under vacuum for 30 minutes, rinsed twice with distilled water, and then washed with washing buffer (10 mM Tris-HCl pH 7.2 and 50 mM KCl). Subsequently, the samples were incubated with 50 mM H2DCFDA staining solution in the dark at room temperature for 10 minutes, and excess dye was removed twice with distilled water. All samples were examined using a confocal microscope (Olympus FV3000, Japan) with excitation at 488 nm and emission at 530 nm. To compare fluorescence intensity, all parameter axes from different experimental conditions were fixed, and analysis was performed under the confocal microscope using the same settings. The fluorescence intensity was calculated using ImageJ software.

[0145] This experiment examined the reactive oxygen species (ROS) levels in the hairy roots of MsGADP2 transgenic alfalfa, EV, and RNAi transgenic alfalfa by staining with H2DCFDA (cytosol redox state) after salt treatment. The results showed that ROS levels were low and showed little difference in all untreated alfalfa hairy roots. After salt treatment, compared to the wild-type control, less ROS accumulation was detected in MsGADP2-overexpressing hairy roots, while ROS accumulation was significantly increased in RNAi transgenic alfalfa hairy roots. Figure 13 These results suggest that overexpression of MsGADP2 may have the function of regulating intracellular ROS levels and preventing plant roots from being damaged under salt stress.

[0146] This experiment, using reactive oxygen species (ROS) staining, revealed that overexpression of MsGADP2 reduced ROS accumulation in roots. Therefore, the potential role of MsGADP2 in maintaining ROS homeostasis was further evaluated by exogenous application of H2O2. Firstly, in the yeast heterologous expression experiment, the growth performance of transgenic yeast cells and untransgenic empty yeast cells was similar under control conditions. After exposure to 15 mM H2O2 stress, the growth of transgenic yeast cells was significantly better than that of untransformed empty yeast cells. This preliminarily verified that overexpression of MsGADP2 reduced ROS accumulation in roots. Secondly, in this experiment, root length measurements on salt-treated plates with exogenous H2O2 showed that lateral roots of both EV and OE were inhibited compared to the control. However, in salt-treated plates with exogenous reduced glutathione (GSH), root length measurements showed that lateral roots lacking MsGADP2 function recovered growth, with no significant difference in phenotype compared to the control. Figure 14 This indicates that under salt stress, H2O2 is produced and accumulates, leading to oxidative stress in plants and reducing their salt tolerance, while reducing H2O2 levels helps improve their salt tolerance. The results suggest that overexpression of MsGADP2 may protect cells from oxidative damage by regulating intracellular ROS homeostasis, and highlight the importance of maintaining intracellular ROS homeostasis for lateral root growth and development under salt stress.

[0147] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A Medicago sativa MsGADP2 protein, characterized in that, The amino acid sequence of which is shown as SEQ ID NO.

2.

2. A nucleic acid molecule, characterized in that, The nucleotide sequence of which is shown as SEQ ID NO.

1.

3. The nucleic acid molecule of claim 2, wherein, The nucleotide sequence of which is shown as SEQ ID NO.

1.

4. A recombinant vector, characterized in that, The nucleotide sequence of which is shown as SEQ ID NO.

1.

5. A recombinant bacterium, characterized in that, The nucleotide sequence of which is shown as SEQ ID NO.

1.

6. The application of the alfalfa MsGADP2 protein and its encoding gene in improving the salt stress resistance of plants, wherein the plants are Arabidopsis thaliana and Medicago sativa.

7. Use according to claim 6, characterized in that, The application comprises regulating the lateral root development of plants under salt stress.

8. A method for improving the salt stress tolerance of a plant, characterized in that, The application comprises introducing the nucleotide molecule of claim 2 or 3 into the genome of a target plant to obtain a transgenic plant; The plants are Arabidopsis thaliana and Medicago sativa.

9. A method of breeding plants, characterized in that, The application comprises: overexpressing the gene of the alfalfa MsGADP2 protein of claim 1 in plants to improve the salt stress resistance of the plants; The plants are Arabidopsis thaliana and Medicago sativa.

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