Medicago sativa MsSNAT1 gene and application of medicago sativa MsSNAT1 gene in regulation and control of plant salt tolerance

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a medicago sativa MsSNAT1 gene and application of the medicago sativa MsSNAT1 gene in regulation and control of plant salt tolerance. The nucleotide sequence of the medicago sativa MsSNAT1 gene is as shown in SEQ ID NO. 1. Experimental results show that overexpression of the MsSNAT1 gene enhances the stress resistance of medicago sativa. The overexpression of the MsSNAT1 gene promotes the increase of the content of endogenous melatonin and increases the expression of the melatonin synthetase gene under salt stress. Compared with a wild type, the antioxidant enzyme activity and osmoregulation substance increase trend of the transgenic line under salt and drought stress is more obvious, the MDA content is lower, and the negative influence is smaller. In addition, the content of endogenous melatonin in a transgenic line is increased, and the endogenous melatonin and exogenous ABA act together, so that the oxidation resistance and osmotic regulation capacity of the plant are enhanced, and the stress resistance of the plant is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and particularly relates to the alfalfa MsSNAT1 gene and its application in regulating plant salt tolerance. Background Art

[0002] Alfalfa (Medicago sativa L.) is one of the important forage crops in the world and has extremely rich nutritional value. However, since most of its growth areas are in saline-alkali desert areas, the yield and quality of alfalfa are severely affected. Melatonin is a pleiotropic and multi-active molecule that can promote plant growth, protect plants from adversity, and regulate flowering and fruit ripening by regulating the redox network and the intervention of other plant hormones. SNAT is a key gene in the melatonin synthesis process and plays an important role in the melatonin synthesis process. By applying exogenous melatonin and the genetic engineering regulation technology of the melatonin synthesis pathway to increase the endogenous melatonin content, the damage of salt stress to alfalfa can be effectively avoided. Therefore, the research on the key genes of the melatonin synthesis molecular mechanism in alfalfa is of great significance. In the prior art, there have been a large number of reports on the regulation of salt tolerance genes to enhance plant salt tolerance. The SNAT gene has been cloned in some plants, but there is little research on the function of the MsSNAT gene in alfalfa. Summary of the Invention

[0003] Aiming at the above deficiencies in the prior art, the purpose of the present invention is to provide the alfalfa MsSNAT1 gene and its application in regulating plant salt tolerance.

[0004] In order to achieve the above invention purpose, the technical solution adopted by the present invention is as follows:

[0005] In the first aspect, the alfalfa MsSNAT1 gene is provided, and its nucleotide sequence is as shown in SEQ ID NO.1.

[0006] In the second aspect, a transgenic vector containing the alfalfa MsSNAT1 gene is provided.

[0007] In the third aspect, a recombinant strain or recombinant cell containing the alfalfa MsSNAT1 gene is provided.

[0008] In the fourth aspect, the application of overexpression of the alfalfa MsSNAT1 gene in improving plant salt tolerance is provided.

[0009] In the fifth aspect, the application of the alfalfa MsSNAT1 gene in improving the resistance quality of alfalfa and breeding excellent salt-tolerant alfalfa varieties is provided.

[0010] The beneficial effects of the present invention are as follows:

[0011] The present invention cloned the MsSNAT1 gene related to salt tolerance from alfalfa, constructed a recombinant vector of the MsSNAT1 gene, and verified the function of the MsSNAT1 gene in improving the salt tolerance of plants. The present invention can be used to improve the salt tolerance of alfalfa and create alfalfa germplasm materials with strong salt tolerance. Description of the Drawings

[0012] Figure 1 It is a diagram of the pCE2-TA / Blunt-Zero vector;

[0013] Figure 2 It is a skeleton diagram of the 3-2 pCAMBIA2300s vector;

[0014] Figure 3 It is a gel electrophoresis diagram of total RNA of alfalfa;

[0015] Figure 4 It is the amplification of the MsSNAT1 gene of alfalfa;

[0016] Figure 5 It is a gel electrophoresis diagram for the identification of positive monoclonal clones;

[0017] Figure 6 It is the sequence alignment of the MsSNAT gene;

[0018] Figure 7 It is the analysis of the conserved domain of MsSNAT1;

[0019] Figure 8 It is the amino acid sequence alignment of homologous species;

[0020] Figure 9 It is the SNAT phylogenetic tree;

[0021] Figure 10 It is the PCR of the target gene;

[0022] Figure 11 It is the double digestion verification; 1: electrophoresis of the original plasmid 2: electrophoresis of the recombinant plasmid M: DL10000;

[0023] Figure 12 It is the bacterial liquid PCR;

[0024] Figure 13 It is the sequencing result comparison diagram;

[0025] Figure 14 It is the tissue culture process of transgenic MsSNAT1 alfalfa; A. Co-culture; B. Screening for 3 days; C. Screening for 7 days; D. Differentiation; E, F. Rooting; G. Seedling formation;

[0026] Figure 15 It is the identification of positive seedlings of transgenic MsSNAT1 alfalfa;

[0027] Figure 16 are the contents of endogenous melatonin in the control strain and transgenic strain under normal culture conditions;

[0028] Figure 17 are the phenotypic comparisons between wild-type alfalfa and transgenic alfalfa under salt stress, drought stress, and ABA treatment;

[0029] Figure 18 are the plant heights, stem diameters, root lengths, and branch numbers of wild-type and transgenic alfalfa under different treatments; among them, A is the plant height; B is the stem diameter; C is the root length; D is the branch number;

[0030] Figure 19 are the fresh weights and dry weights of wild-type and transgenic alfalfa under different treatments; among them, A is the fresh weight; B is the dry weight;

[0031] Figure 20 are the relative chlorophyll contents of wild-type and transgenic alfalfa under different treatments;

[0032] Figure 21 are the antioxidant enzyme activities and MDA contents of wild-type and transgenic alfalfa under different treatments; among them, A is SOD; B is POD; C is CAT; D is APX; E is MDA;

[0033] Figure 22 are the proline contents of wild-type and transgenic alfalfa under different treatments;

[0034] Figure 23 are the expression levels of antioxidant enzyme genes in wild-type and transgenic alfalfa under different treatments; among them, A is MsSOD; B is MsCatalase4; C is MsAPX; D is MspxdC. Detailed implementation manners

[0035] The following describes the detailed implementation manners of the present invention to facilitate those skilled in the art of this technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the detailed implementation manners. For those of ordinary skill in the art of this technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions created using the concept of the present invention are within the scope of protection.

[0036] Examples

[0037] 1. Cloning of alfalfa MsSNAT1 gene and construction of plant expression vector

[0038] 1.1 Test materials

[0039] 1.1.1 Plant materials, strains, and vector backbones

[0040] Taking Medicago sativa cv. Zhongmu No. 1 planted in the artificial climate chamber of the School of Ecology and Environment, Ningxia University as the material, the freshly picked leaves were quickly placed in liquid nitrogen and then stored in a -80 °C ultra-low temperature freezer.

[0041] The plant expression vector pCAMBIA2300s was stored in this laboratory. Escherichia coli TOP 10 (product number: WH0190) was purchased from Shanghai Sangon Biotech Co., Ltd.

[0042] 1.1.2 Test reagents

[0043] The polysaccharide and polyphenol plant total RNA extraction kit used for gene cloning was purchased from TianGen (product number: DP441); the reverse transcription kit HisScript 1st Strand cDNA was purchased from Vazyme (product number: R312-01); TaKaRa Taq TM was purchased from Takara (product number: R001A); the DNA purification gel recovery kit was purchased from TianGen (product number: DP214); the T cloning vector 5minTA / Blunt-Zero Cloning Kit was purchased from Vazyme (product number: C601); the competent cells of Escherichia coli TOP 10 were purchased from Shanghai Sangon; the plasmid miniprep kit was purchased from TianGen (product number: DP103); T4 DNA Ligase was purchased from TaKaRa (product number: 2011A); the restriction endonucleases BamH I / sal I were purchased from TaKaRa; 10×Cutsmart Buffer was purchased from Beijing BioLabo Technology Co., Ltd. (product number: SV 0827-DIQ); DL 2000 DNA Maker was purchased from Vazyme (product number: MD101); kanamycin (Kan) and rifampicin (Rif) were purchased from Shanghai Sangon Biotech Co., Ltd.

[0044] 1.1.3 Experimental instruments and equipment

[0045] Electrophoresis apparatus (DYY-6C, Shanghai Danding International Trade Co., Ltd.), pipette (Eppendorf, Germany), PCR gel imaging instrument (IAS-500, Fuyue Biotechnology Co., Ltd.), constant temperature incubator (DNP-9052, Qingdao Mingbo Environmental Protection Technology Co., Ltd.), temperature-controlled shaker (HYM-103B, Qingdao Mingbo Environmental Protection Technology Co., Ltd.), autoclave (LDZX-30KBS, Suzhou Aopu Experimental Equipment Co., Ltd.), digital display constant temperature water bath (HH-6, Shanghai Gaozhi Precision Instrument Co., Ltd.), laminar flow hood (AIPTECH, Suzhou Yabao Purification Technology Co., Ltd.), low temperature centrifuge (Eppendorf, Germany).

[0046] 1.1.4 Preparation of Culture Media and Antibiotics

[0047] The reagents were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Preparation of LB solid medium (Table 1-1): Weigh 2.5 g of yeast powder, 5 g of peptone, 5 g of NaCl, and 3.5 g of agar powder in sequence using a balance, and place them in an Erlenmeyer flask with a specification of 500 ml. Add distilled water to the Erlenmeyer flask and make the volume up to 500 ml, then adjust the pH to 7.0. Autoclave at 121 °C for 30 min. When the temperature cools to about 55 °C, add the corresponding antibiotics in a laminar flow hood, and then slowly and evenly pour the medium into the autoclaved glass petri dishes. The amount poured into each dish is about 15 ml. After cooling and solidifying, it is ready for use.

[0048] Table 1-1 LB Medium Formula

[0049]

[0050]

[0051] Kan(50 mg·mL -1 ): Dissolve 0.5 g of Kan in a small amount of sterile water, make the volume up to 10 mL, and dispense and store in 1.5 mL sterilized centrifuge tubes at -20 °C.

[0052] Rif(50 mg·mL -1 ): Dissolve 0.5 g of Rif in a small amount of methanol in a light-proof environment, make the volume up to 10 mL, and dispense and store in 1.5 mL sterilized centrifuge tubes at -20 °C.

[0053] 1.1.5 Preparation of Agarose Gel

[0054] 1×TAE: Take 20 mL of 50×TAE electrophoresis buffer and make the volume up to 1 L

[0055] Take 40 mL of the above solution, add 0.4 g of agarose, heat and dissolve it thoroughly. After cooling, add 4 μL of nucleic acid dye Gelred, shake well and pour it onto the plate with a comb inserted.

[0056] 0.5×TAE: Take 10 mL of 50×TAE electrophoresis buffer and make the volume up to 1 L. It is used for RNA electrophoresis.

[0057] 1.2 Experimental Methods

[0058] 1.2.1 Extraction of Alfalfa RNA

[0059] Take out the alfalfa seedling leaves from an ultra-low temperature refrigerator at -80°C for RNA extraction. Weigh 10 mg of the leaves with a balance and put them into a sterilized mortar. Quickly add liquid nitrogen and grind them into a fine powder. Collect the well-ground powder into a 1.5 mL centrifuge tube and extract it using a polysaccharide and polyphenol plant total RNA extraction kit (TIANGEN). The method is as follows:

[0060] ① Add 600 μL of lysis buffer SL (β-mercaptoethanol has been added before use) to the centrifuge tube containing plant tissue, immediately vortex and mix well, and centrifuge at 12,000 rpm for 2 min at 4°C;

[0061] ② Transfer the supernatant to the filter column CS (the filter column CS is placed in the collection tube), centrifuge at 12,000 rpm for 2 min at 4°C, and carefully aspirate the supernatant in the collection tube into a new ribonuclease-free centrifuge tube (try to avoid the pipette tip touching the cell debris precipitate in the collection tube),

[0062] ③ Carefully add 240 μL of absolute ethanol to the centrifuge tube from the previous step, gently pipette and mix well, transfer the resulting mixture into the adsorption column CR3, centrifuge at 12,000 rpm for 1 min at 4°C, then pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube;

[0063] ④ Add 350 μL of protein removal solution RW1 to the CR3 from the previous step, centrifuge at 12,000 rpm for 1 min at 4°C, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube;

[0064] ⑤ Add 500 μL of washing solution RW to the adsorption column CR3, centrifuge at 12,000 rpm for 1 min at 4°C, pour out the waste liquid in the collection tube, put the adsorption column CR3 back into the collection tube, repeat this step twice, and then centrifuge at 12,000 rpm for 2 min to thoroughly elute the RNA.

[0065] ⑥ Place the adsorption column CR3 into a new ribonuclease-free centrifuge tube, suspend and add 40 μL of RNase-Free ddH 2 O to the middle part of the adsorption membrane, let it stand at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min at 4°C to obtain the RNA solution;

[0066] Use agarose gel electrophoresis with a gel concentration of 1.2% to detect the integrity of the bands of the obtained RNA solution. The electrophoresis conditions are: 0.5×TAE electrophoresis buffer; 150 V, 15 min. Use a ultra-micro ultraviolet spectrophotometer to read the OD260 / OD280 ratio to check for protein contamination. Take 2.5 μL of the RNA solution and use RNase-Free ddH 2Zero the spectrophotometer, take the diluted solution for OD260 and OD280 measurements, and calculate the RNA concentration according to the following formula:

[0067] Final concentration (ng / μL) = (OD260) × (dilution factor) × 40

[0068] 1.2.2 Reverse transcription of total RNA from alfalfa into cDNA

[0069] Use the total RNA from alfalfa extracted above as a template, and utilize the reverse transcription kit HisScript 1stStrand cDNA (Vazyme) for cDNA synthesis. The reverse transcription operation procedure is as follows:

[0070] ① RNA template denaturation: This step helps to open the RNA secondary structure and can greatly improve the yield of the first-strand cDNA. After adding the following reagents to an RNase-Free tube (Table 1-2), heat it in a 65°C water bath for 5 min, then quickly place it on ice for rapid cooling and let it stand for 2 min.

[0071] Table 1-2 RNA template denaturation reaction system

[0072]

[0073]

[0074] ② Genomic DNA removal: After adding the following reagents (Table 1-3), gently pipette and mix well, and react at 42°C for 2 min.

[0075] Table 1-3 Genomic DNA removal reaction system

[0076]

[0077] ③ Prepare the first-strand cDNA synthesis reaction solution: After adding the following reagents (Table 1-4), gently pipette and mix well. Place the mixture in a PCR instrument for reaction. The temperature settings of the PCR instrument are: 25°C for 2 min, 37°C for 45 min, 85°C for 5 sec. Aliquot the cDNA product into sterile PCR tubes and store it in a -20°C refrigerator.

[0078] Table 1-4 First-strand cDNA synthesis reaction solution system

[0079]

[0080] 1.2.3 Primer design and cloning

[0081] With reference to the annotation results of the transcriptome data of alfalfa measured in the laboratory, the SNAT gene was screened out, and the nucleotide sequence of the related gene was sorted out, and this gene sequence was used as the reference sequence. According to the alfalfa genome sequence published on NC, BLAST obtained the sequence information, and specific primers were designed in the 3'UTR and 5'UTR regions of the sequence, mainly using SnapGene software, Primer Premier5.0 auxiliary inspection, and synthesized by BGI Biotechnology Co., Ltd. The full-length CDS sequence of the MsSNAT gene was cloned using PCR technology. The principles of primer design are as follows:

[0082] ①The 3' end should avoid high GC / high AT content areas as much as possible;

[0083] ② The primer Tm is adjusted close to 60℃, and the Tm value of the forward and reverse primers should not differ by more than 1℃;

[0084] ③GC content is 40%-60%, and the four bases are evenly distributed in the primers;

[0085] ④Try to avoid continuous structures starting with G / C or A / T;

[0086] ⑤Try to avoid the presence of reverse repeat sequences or self-complementary sequences greater than 3bp;

[0087] 1.2.3.1 Target fragment amplification

[0088] Utilize TaKaRa Taq TM (TaKaRa) kit was used to amplify the target fragment, and alfalfa cDNA was used as a template, upstream primer F and downstream primer R were used to amplify the target fragment. A 20μL system was used for the first amplification, and the amplified product was tested for clarity and correctness of the band using 1% agarose gel electrophoresis. After confirming that the band was correct, the test was repeated with a 50μL system, and the fragment of the correct size was purified and recovered. The 50μL amplification system is shown in Table 1-5. The PCR reaction program is: 94℃5min; 94℃10s; 55℃30s; 72℃1min; 72℃7min.

[0089] Table 1-5 Gene amplification reaction system

[0090]

[0091] 1.2.3.2 Gel purification and recovery of target DNA fragment

[0092] Cut the agarose gel with the correct size and clear bands obtained in the previous step with a gel cutter. Aim the gel cutter at the DNA band of the agarose gel and press down quickly and accurately to cut it. Put it into a clean centrifuge tube and weigh it. The specific steps are as follows:

[0093] ①Column equilibration: Place the Spin Columns CB2 into the collection tube, add 500 μL of equilibration buffer BL, centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and then place the adsorption column back into the collection tube again;

[0094] ②Agarose gel block: Add an equal amount of Buffer PC to the agarose gel block, then place it in a 50 °C water bath for about 10 min, and gently invert the centrifuge tube up and down repeatedly to fully dissolve the gel block;

[0095] ③Loading the solution into the purification column CB2: Add the solution obtained in the above steps to a Spin Columns CB2 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and then place the SpinColumns CB2 back into the collection tube again;

[0096] ④Rinsing the adsorption column: Add 600 μL of wash buffer PW to the Spin Columns CB2, let it stand for 2 min, then centrifuge for 1 min, pour out the waste liquid and place the Spin Columns CB2 back into the collection tube. Repeat this step twice;

[0097] ⑤Washing and drying the adsorption column: Place the Spin Columns CB2 back into the collection tube, centrifuge at 12,000 rpm for 2 min to wash away the wash buffer. Then place the adsorption column in the laminar flow hood for 5 min to dry it;

[0098] ⑥Obtaining DNA: Place the Spin Columns CB2 into a clean centrifuge tube, suspend and add 40 μL of ddH 2 O to the middle of the adsorption membrane, place it in the laminar flow hood for 2 min, and then centrifuge at 12,000 rpm for 2 min to collect the DNA solution;

[0099] ⑦Storing DNA: Store the obtained DNA solution at -20 °C for future use.

[0100] 1.2.3.3 Ligation of the T-cloning vector and the recovered product

[0101] Ligate the purified target fragment to the TA-Zero Cloning vector. The ligation system is shown in Table 1-6. Gently flick the bottom of the PCR tube to mix the reaction solution, and centrifuge at low speed instantaneously to collect the reaction solution at the bottom of the tube. Set the PCR temperature to 25 °C for 30 min. After the reaction, place the centrifuge tube on ice. The skeletal diagram of the pCE2-TA-Zero vector is Figure 1.

[0102] Table 1-6 Ligation system of the cloning vector

[0103]

[0104] 1.2.3.4 Transformation of the ligation product into competent E. coli TOP 10 cells

[0105] Select competent E. coli TOP 10 cells (TianDZ). Take out the competent cells from the -80 °C refrigerator and place them on an ice box. When they melt into a mixture of ice and water, add all 5 μL of the ligation product to the competent cell suspension, gently flick the tube wall to mix evenly, then let it stand on the ice box for 30 min. After standing, perform heat shock in a 42 °C water bath for 90 sec, then quickly transfer the tube to the ice box and perform an ice bath for 3 min. In a laminar flow hood, use a pipette to add 200 μL of sterile LB liquid to the centrifuge tube, gently shake to mix evenly, then place the centrifuge tube on a 37 °C temperature-controlled shaker and shake at 150 rpm for 45 min to allow the bacteria to recover. After the bacteria recover, aspirate 40 μL of the transformed competent cells and add them to the LB solid medium containing kanamycin and rifampicin using a pipette. Use a sterile pipette tip to bend it and gently spread the cells evenly on the plate. After spreading the plate, place it in the laminar flow hood until the bacterial liquid is absorbed. Invert the plate and place it in a 37 °C constant temperature incubator to culture overnight.

[0106] 1.2.3.5 Screening and identification of positive colonies

[0107] In the laminar flow hood, use a sterile pipette tip to pick monoclonal colonies on the LB plate with a size of 2 - 3 mm and add them to 20 μL of ddH 2 O and pipette to mix evenly. Aspirate 5 μL from the mixed bacterial liquid as the template for bacterial liquid PCR. Add the reagents in Table 1 - 7 to the PCR tube in sequence. After all the samples are added, gently flick to mix evenly, and centrifuge at 6000 rpm for 15 sec to collect the reaction components at the bottom of the tube. Use the universal primers M13F and M13R on the vector to screen for positive clones. This step is performed on ice throughout the process. Add the remaining bacterial liquid to 800 μL of LB liquid medium containing 50 mg / mL of kanamycin for bacterial liquid expansion, shake overnight at 37 °C, and send the corresponding bacterial liquid of the positive clones that have been screened to Shanghai Sangon Biotech Co., Ltd. for sequencing. Extract the plasmid of the bacterial liquid and store it at -80 °C.

[0108] Table 1 - 7 Reaction system for screening positive colonies

[0109]

[0110] Setting of the PCR reaction thermal cycling program:

[0111]

[0112]

[0113] 1.2.4 Sequencing Results and Bioinformatics Analysis

[0114] The sequencing results were aligned and analyzed with the full-length CDS sequence of MsSNAT using the DNAMAN software. Cross-species sequence comparison was analyzed using DNAMAN, and a phylogenetic tree was constructed using the Neighbor-Joining method of the MEGA software.

[0115] 1.2.5 Construction of the MsSNAT1 Gene Recombinant Overexpression Vector

[0116] 1.2.5.1 Extraction of Vector Plasmid

[0117] Escherichia coli containing the pCAMBIA2300s vector stored in a glycerol tube was taken out from the -80 °C refrigerator and placed on an ice box to melt. 1 μL of the bacterial solution was pipetted into 1 mL of sterile ddH 2 O for dilution and mixed well by pipetting with a sterile pipette tip. 30 μL of the bacterial solution was evenly spread on an LB solid medium containing 50 μg / mL kanamycin, and the culture plate was inverted and incubated overnight in a 37 °C constant temperature incubator. Single colonies with a size of 2 - 3 mm were picked in a sterile workbench and added to an LB liquid medium containing 50 μg / mL kanamycin and incubated overnight. Centrifuge at 12000 rpm for 1 min to collect the bacteria at the bottom of the centrifuge tube; the plasmid was extracted using a plasmid mini-prep kit (TIANGEN):

[0118] ① Column equilibration step: Add 500 μL of equilibration buffer BL to the adsorption column Spin Columns CP3, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the Collection Tubes, and place the adsorption column back into the Collection Tubes;

[0119] ② Transfer 1 mL of the overnight culture broth to a centrifuge tube, centrifuge at 12000 rpm for 1 min, discard the supernatant, add 250 μL of P1 solution to the centrifuge tube containing the bacterial solution, and mix the bacterial solution and the solution well with a pipette;

[0120] ③ Add 250 μL of P2 solution to the above centrifuge tube, gently invert the tube to mix the contents inside the tube and completely dissolve the bacterial solution. The time for the bacterial solution to change from turbid to clear and viscous is less than 5 min to avoid damaging the plasmid.

[0121] ④ Add 350 μL of P3 solution to the above centrifuge tube, gently invert up and down several times until a white flocculent precipitate appears, and centrifuge at 12000 rpm for 10 min;

[0122] ⑤ Transfer the supernatant collected in the previous step to the adsorption column CP3 using a pipette, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and transfer the adsorption column CP3 to a collection tube;

[0123] ⑥ Add 600 μL of wash buffer PW to the adsorption column CP3, centrifuge at 12,000 rpm for 1 min, discard the waste liquid and transfer the adsorption column CP3 to a collection tube. Repeat this step twice; then centrifuge at 12,000 rpm for 2 min to thoroughly wash away the wash buffer PW as much as possible;

[0124] ⑦ Place the adsorption column CP3 in a clean centrifuge tube, add 60 μL of ddH 2 O to the middle part of the adsorption membrane, place it in a laminar flow hood for 2 min, and finally centrifuge at 12,000 rpm for 2 min to collect the plasmid solution into the centrifuge tube. The plasmid is stored in a -20 °C refrigerator for later use.

[0125] 1.2.5.2 Preparation and Amplification of the Target Fragment

[0126] Perform a multiple cloning site analysis on the plasmid map of the pCAMBIA2300s vector ( Figure 2 ), and at the same time analyze the restriction enzyme sites within the cloned MsSNAT gene. To prevent the restriction enzyme sites from cutting the MsSNAT1 gene, select the ones that are present on the vector but not within the MsSNAT1 gene. Therefore, the restriction enzyme sites are selected as: BamH I / sal I. According to the primer design principle, introduce the BamH I (upstream) and sal I (downstream) restriction enzyme sites into the primers. Use SnapGene to design primers F1 and R1 for amplifying the full-length CDS sequence of the Medicago sativa MsSNAT1 gene cDNA. The primers are synthesized by BGI Shenzhen Co., Ltd.

[0127] Use the T-cloning vector as the amplification template to amplify the target fragment with restriction enzyme sites. The specific experimental method is the same as in 1.2.3.

[0128] 1.2.5.3 Double Digestion

[0129] Use the restriction endonucleases BamH I and sal I to cut the target gene plasmid and the pCAMBIA2300s vector. The double digestion reaction system is shown in Table 1-8, and the double digestion reaction conditions are: the digestion reaction is carried out in a 37 °C constant temperature water bath for 15 - 25 min. The double digestion reaction identification reaction system is shown in Table 1-9. After the digestion reaction, the reaction products need to be detected by agarose gel electrophoresis, and the vector is determined whether it is cut by observing the band positions.

[0130] Table 1-8 Vector Plasmid Digestion System

[0131]

[0132] Table 1-9 Double digestion identification system

[0133]

[0134]

[0135] 1.2.5.4 Ligation to obtain the recombinant plasmid pCAMBIA2300s-MsSNAT1

[0136] Purify and recover the products of double digestion of MsSNAT1 and pCAMBIA2300s. Add the gel-purified products and T4 ligase into a PCR tube, and gently pipette and mix well. After transient centrifugation, place it at 16 °C for overnight ligation. The recombinant reaction system is shown in Table 1-10.

[0137] Table 1-10 Recombinant reaction system

[0138]

[0139] 1.2.5.5 Transformation of the recombinant plasmid product into competent cells of Escherichia coli E.coli TOP 10

[0140] Transform the recombinant plasmid into competent Escherichia coli cells using the same method as in 1.2.3.4. Spread the transformation solution on a solid LB medium containing 50 mg / ml Kan and incubate it upside down in a 37 °C constant temperature incubator overnight.

[0141] 1.2.5.6 Identification and sequencing of positive monoclonal of the recombinant expression vector

[0142] Use the enzyme digestion primers of the MsSNAT1 gene as the primers for bacterial liquid PCR. The primer sequences are shown in Table 1-11. Expand the bacterial liquid that has been correctly detected by PCR gel electrophoresis, place it in an LB liquid medium containing 50 mg / ml Kan and shake it overnight, and then send the bacterial liquid to a biological company for sequencing. The experimental steps are shown in 1.2.3.5.

[0143] 1.3 Results and analysis

[0144] 1.3.1 Identification of the quality of total RNA from alfalfa

[0145] Extract the RNA of Medicago sativa cv. Zhongmu No.1 using the TianGen plant RNA extraction kit as the template for gene amplification. The RNA gel electrophoresis pattern shows ( Figure 3), the extracted alfalfa RNA was in good integrity, with clear and bright bands of 18S and 28S. To further determine the quality of this RNA, ultraviolet spectrophotometer quantitative analysis was carried out on it. The results showed that the OD of most samples was measured at a concentration of 463 μL / mL, and the OD 260 / 280 ratio was between 1.9 and 2.0, indicating no protein contamination. Therefore, it can be used as a template for gene cloning. Take the RNA that meets the quality requirements and perform reverse transcription according to the 1 μg / 10 μL system to obtain the first strand of cDNA. The concentration after reverse transcription into cDNA is 1337 μg·mL -1 .

[0146] 1.3.2 Obtaining the alfalfa MsSNAT1 gene

[0147] 1.3.2.1 Primer design and gene cloning

[0148] Based on the alfalfa genome sequence published on NCBI, sequence information was obtained by BLAST. According to this sequence information, using SnapGene software as the main tool and Primer Premier5.0 for auxiliary verification, upstream and downstream primers were designed from the UTR ends of the sequence for cloning respectively. The primer sequences are as follows (Table 1-11). Using Zhongmu No. 1 as the template, the PCR amplification products were detected by 1% agarose gel electrophoresis. The results are as Figure 4 , the target gene band was clear and bright, and the gene sequence with a fragment size of about 600 bp. It was sent to the company for sequencing. After sequencing and comparison, the band size was correct. The results showed that the cloned target fragment sequence of the gene was consistent with the alfalfa genome data sequence, proving that the MsSNAT1 gene was successfully cloned.

[0149] Table 1-11 Primers for gene cloning

[0150]

[0151] 1.3.2.2 Identification of positive colonies of the cloning vector and sequence acquisition

[0152] According to the verification by agarose gel electrophoresis, a band with the correct target fragment size was obtained, and DNA purification and recovery were carried out. The recovered DNA was ligated with TA-Zero Cloning and heat-shock transformed into E. coli TOP 10 competent cells. The transformation method was as described in 1.2.3.4. Take 30 μL of the bacterial solution and gently spread it on the prepared LB plate containing kanamycin, and incubate overnight at 37 °C. Single colonies were picked, and colony PCR identification was carried out using universal primers (M13F and M13R). The positive colonies successfully transformed into E. coli TOP 10 were amplified and cultured. 5 monoclonal colonies appeared in the experiment, and these 5 monoclonal colonies were sent to the biological company for sequencing( Figure 5) Finally, the CDS sequence of the alfalfa MsSNAT gene was obtained, with a length of 588 bp.

[0153] 1.3.3 Sequencing results

[0154] The sequencing results were put into the DNAMAN software and compared with the full-length CDS sequence of the target gene MsSNAT as Figure 6 . The homology of the sequence reached 99.32%, which proved that the sequence was cloned and had a length of 588 bp, named MsSNAT1.

[0155] 1.3.4 Bioinformatics analysis

[0156] 1.3.4.1 Conserved domain analysis

[0157] The NCBI was used to analyze the domain of the coding region of the alfalfa MsSNAT1 protein, and the results were as Figure 7 . MsSNAT1 belongs to a member of the NAT-SF super family, and the domain is complete.

[0158] 1.3.4.2 Homology and phylogenetic tree analysis of the MsSNAT1 gene

[0159] The BLAST function of NCBI was used to obtain homologous species of the alfalfa MSNAT1 protein. Ten sequences with relatively high homology were selected, the sequences were downloaded and compared using the DNAMAN software ( Figure 8)。The SNAT homologous species and their accession numbers are as follows: chickpea CaSNAT (XP_004490320.1), pea PsSNAT (XP_050905241.1), broad bean VfSNAT (CAI8595604.1), subterranean clover TsSNAT (GAU51507.1), red bartsia TpSNAT (XP_045819016.1), sesbania cannabina SbSNAT (KAJ1412052.1), azuki bean VaSNAT (XP_017434999.1), white lupin LaSNAT (KAE9595708.1), mung bean VrSNAT (XP_014517051.1), soybean GmSNAT (XP_003518810.1). Through sequence alignment, it was found that: the homology of Medicago sativa SNAT1 with chickpea CaSNAT, pea PsSNAT, and broad bean VfSNAT is relatively high, reaching 82.38%, 82.9%, and 82.38% respectively; the homology with subterranean clover TsSNAT, red bartsia TpSNAT, and sesbania cannabina SbSNAT is 79.79%, 78.24%, and 78.38% respectively. Using the MEGA7 software, the Neighbor-Joining method was used to analyze the phylogenetic tree of the Medicago sativa MsSNAT1 protein sequence and related species ( Figure 9 )。

[0160] 1.3.5 Construction of recombinant overexpression vector

[0161] 1.3.5.1 Primer design and amplification

[0162] Using the T recombinant plasmid as a template and F1 and R2 as primers for amplification of the target fragment. The results are as Figure 10 , lanes 1, 2, and 3 are the amplified target genes, with a size of approximately 600 bp, indicating that the target gene was obtained.

[0163] Table 1-12 Primer design for construction of overexpression vector

[0164]

[0165] 1.3.5.2 Double digestion verification

[0166] The agarose gel containing the target gene MsSNAT1 and digested pCAMBIA2300s was cut and recovered, and the purified products were ligated with T4 ligase to obtain the recombinant plasmid. The double digestion identification results are as in 11, lane 1 is the electrophoresis of the original plasmid, and lane 2 is the electrophoresis of the recombinant plasmid. According to the band positions, it indicates that the digestion was successful.

[0167] 1.3.5.3 Bacterial liquid PCR

[0168] Use the MsSNAT1 gene digestion primer as the colony PCR primer. Expand the colony solution that has been correctly detected by PCR gel electrophoresis, put it into LB liquid medium containing 50 mg / ml Kan, shake it overnight, and then send the colony solution to a biological company for sequencing. The PCR results are as Figure 12 .

[0169] 1.3.5.4 Sequencing result analysis

[0170] Compare the sequencing results with the measured CDS sequence at the same time, and the analysis results are as Figure 13 , with a homology of 99.83%, proving that the gene has been successfully cloned into the plant expression vector, and the recombinant vector is named pCAMBIA2300s-MsSNAT1.

[0171] 2. Stress resistance evaluation of MsSNAT1-OE transgenic alfalfa

[0172] 2.1 Genetic transformation and molecular detection of transgenic MsSNAT1 alfalfa

[0173] 2.1.1 Genetic transformation of transgenic MsSNAT1 alfalfa

[0174] The genetic transformation of alfalfa was carried out by the Agrobacterium-mediated method. The specific steps are as follows:

[0175] (1) Bacterial shaking: Take out the Agrobacterium tumefaciens solution of the pCAMBIA2300s-MsSNAT1 plant overexpression vector from -80 °C, pipette 10 μl and spread it on an LB solid plate containing KanR (50 mg·L -1 ) and Rif (50 mg·L -1 ), culture it overnight at 28 °C for 60 h. After single colonies grow, pick a single colony and add it to an LB culture solution containing antibiotics, and culture it with shaking at 28 °C and 200 rpm for activation. Pipette 50 μl of the activated Agrobacterium solution from the small flask into 50 ml of LB culture solution containing antibiotics, and culture it with shaking at 28 °C and 200 rpm until the OD600 value is 0.6 - 0.8.

[0176] (2) Leaf cleaning: Collect the leaves of healthy alfalfa 'Zhongmu No. 1', put the leaves into a sterile tissue culture bottle in a laminar flow hood, rinse them with 75% alcohol for 30 s, then wash them three times with distilled water, add 1% mercuric chloride solution, disinfect for 8 min, and rinse with distilled water 4 - 5 times.

[0177] (3) Resuspension: Centrifuge the cultured Agrobacterium solution at 4000 rpm for 15 min. In the laminar flow hood, pour out the supernatant and dilute and resuspend it with the infection solution.

[0178] (4) Infection: Transfer the washed leaves into a sterile tissue culture flask, and add the prepared infection solution. Place it in a desiccator and evacuate for 10 min.

[0179] (5) Drying: Transfer the infected leaves onto a culture dish lined with sterile filter paper in a laminar flow hood for drying.

[0180] (6) Co-culture: Inoculate the infected leaves onto the co-culture medium and culture them in the dark for 2 - 3 d.

[0181] (7) Screening culture: After co-culture, transfer the leaves onto the screening medium and culture them in the dark for about one month.

[0182] (8) Differentiation culture: When bud points appear on the callus in the screening medium, transfer it onto the differentiation medium for continued growth.

[0183] (10) Rooting culture: When young leaves grow out, transfer them to the rooting medium to induce root growth.

[0184] (11) Acclimatization: Transplant the alfalfa seedlings that have grown into seedlings in the rooting medium into flower pots with vermiculite:nutrient soil at a ratio of 3:1. After the transgenic seedlings are acclimatized and grown under high humidity conditions for 3 d, transfer them to normal growth conditions.

[0185] 2.1.2 Molecular detection of transgenic MsSNAT1 alfalfa

[0186] Extract the DNA of wild-type "Zhongmu No. 1" and transgenic alfalfa using the Tiangen DNA extraction kit (DP320). The method is as follows:

[0187] (1) Take 0.1 g of fresh alfalfa leaves, add liquid nitrogen and grind thoroughly. Add 400 μl of buffer LP1 and 6 μl of RNase A, vortex for 1 min, and let it stand at room temperature for 10 min.

[0188] (2) Add 130 μl of buffer LP2, mix well, and vortex for 1 min.

[0189] (3) Centrifuge at 12000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube.

[0190] (4) Add 1.5 times the volume of buffer LP3, and oscillate and mix for 15 sec.

[0191] (5) Add the solution and precipitate obtained in the previous step to the adsorption column CB3, centrifuge at 12000 rpm for 30 sec, and discard the waste liquid.

[0192] (6) Add 600 μl of washing solution PW to CB3, centrifuge at 12000 rpm for 30 sec, and discard the waste liquid. Repeat the previous step.

[0193] (7) Centrifuge at 12,000 rpm for 2 min, pour off the waste liquid, and leave at room temperature for several minutes to thoroughly air-dry the washing solution in the adsorption material.

[0194] (8) Transfer CB3 into a clean centrifuge tube, add 80 μl of elution buffer TE, leave at room temperature for 2 - 5 min, centrifuge at 12,000 rpm for 2 min, and collect the solution into the centrifuge tube.

[0195] The obtained DNA product is stored in a -20 °C refrigerator.

[0196] PCR amplification primers: The upstream primer consists of the vector restriction site, approximately 20 bp of the vector sequence before the restriction site, and approximately 20 bp of the sequence at the 5'-end of the upstream of the target fragment. The downstream primer consists of the downstream vector restriction site, approximately 20 bp of the vector sequence after the restriction site, and approximately 20 bp of the sequence at the 5'-end of the downstream of the target fragment (Table 2-1). The PCR reaction system and procedure are shown in Tables 2-2 and 2-3.

[0197] Table 2-1 PCR primers for identifying transgenic alfalfa

[0198]

[0199] Table 2-2 Reaction system for identifying transgenic alfalfa

[0200]

[0201] Table 2-3 Reaction procedure for identifying transgenic alfalfa

[0202]

[0203]

[0204] 2.2 Determination of endogenous melatonin content and phenotypic analysis of transgenic MsSNAT1 alfalfa

[0205] The experiment used wild-type alfalfa "Zhongmu No. 1" (WT) and transgenic MsSNAT1 alfalfa (OE) under normal culture conditions. Accurately weigh approximately 1 g of fresh plant samples, grind them to powder in liquid nitrogen; add 20 ml of methanol to the powder, sonicate in a water bath for 30 min; centrifuge at 8,000 rpm for 5 min, take the supernatant; dry the organic phase with nitrogen, dissolve it in 200 μl of methanol (0.1% formic acid); filter through a 0.22 μm filter membrane and perform HPLC-MS / MS detection.

[0206] For the determination of growth indicators, the longest branch was measured with a ruler as the plant height; the cross-sectional width of the main branch of the second stem node starting from the bottom of the plant was measured with a vernier caliper as the stem diameter; the above-ground part of the plant was weighed as the fresh weight; after sampling the whole plant, the sample was dried in an oven at 70 °C and then weighed as the dry weight; the plant was carefully dug out of the soil, trying to keep the root system intact, and the root system was rinsed with water to remove attached soil and other impurities. The root system was placed flat on a plate lined with moist filter paper to allow the root system to naturally extend, and a ruler was used to measure the length of the main root and so on.

[0207] 2.3 Study on the stress resistance of transgenic MsSNAT1 alfalfa

[0208] The transgenic lines and the control line "Zhongmu No. 1" were cuttaged and propagated. Select plant materials with good growth conditions, cut branches of the same length as the materials for cuttage propagation. After dipping the lower end of the branches in the rooting powder solution, insert them into flower pots filled with nutrient soil: vermiculite = 1:1 (the nutrient soil and vermiculite should be mixed and watered in advance until completely permeated), and place them in an artificial climate chamber for cultivation. After the cuttings have grown in the artificial climate chamber for 2-3 weeks, transfer them to individual flower pots, taking care not to damage the roots of the seedlings during transfer. The single-seedling cuttings were continued to be placed in the artificial cultivation room for cultivation. After the cuttings have grown for 3-4 weeks, select healthy cuttings without pests and diseases and with consistent growth. The single-seedling cuttings of the wild type (WT) and transgenic lines (OE) were treated with 150 mM NaCl (add 150 ml of NaCl solution every day for 7 days); drought treatment for 7 days; ABA treatment (spray ABA solution on the leaf surface every day for 7 days), with water as the control, and each treatment was repeated three times. Observe the phenotypes of the plants and lines and evaluate their stress resistance.

[0209] 2.4 Results and analysis

[0210] 2.4.1 Genetic transformation and molecular detection of transgenic MsSNAT1 alfalfa

[0211] The tissue culture process of alfalfa is as Figure 14 shown. Using the DNA of transgenic MsSNAT1 alfalfa as the template and WT as the control, PCR amplification reactions were carried out with kanamycin primers and homologous arm primers respectively. The PCR products were detected by 1% agarose gel electrophoresis. Four target fragments were amplified in the PCR reaction and were consistent with the expected length, and no corresponding fragments were amplified from the genomic DNA of WT ( Figure 15 ). The four PCR products were sent to the company for sequencing, and the results were successfully aligned with the MsSNAT1 gene sequence, indicating that 4 positive plants of MsSNAT1 transgenic alfalfa were obtained.

[0212] 2.4.2 Analysis of endogenous melatonin content and phenotype of transgenic MsSNAT1 alfalfa

[0213] To study the changes in melatonin content in alfalfa lines, the endogenous melatonin contents of transgenic lines (OE) and wild-type lines (WT) were determined by HPLC-MS. It was found that under normal nutritional conditions, the melatonin content in WT was only 5.38 ng / ml, which was significantly lower than that in the transgenic line (101.47 ng / ml)( Figure 16 ). The results showed that overexpression of the MsSNAT1 gene significantly increased the endogenous melatonin content in alfalfa.

[0214] Phenotypic identification was carried out on the obtained transgenic lines, and the plant height, number of branches, stem diameter, leaf area, fresh weight, dry weight and root length of CK and OE were statistically analyzed. The results are shown in the table (Table 2-4). Under normal culture conditions, the plant height and root length of OE were significantly 31.64% and 94.77% higher than those of WT, respectively, and the number of branches and leaf area were 14.13% and 33.73% higher than those of WT; while the stem diameter of OE was significantly 37.50% lower than that of WT, and the fresh weight and dry weight were 29.90% and 24.92% lower than those of WT. In summary, under normal culture conditions, the plant height, number of branches, leaf area and root length of transgenic MsSNAT1 alfalfa were higher than those of the control lines, while the stem diameter, fresh weight and dry weight were lower than those of the control lines;

[0215] Table 2-4 Comparison of indexes between control lines and transgenic lines under normal culture conditions

[0216]

[0217] 2.4.3 Study on stress resistance of transgenic MsSNAT1 alfalfa

[0218] Phenotypically, without any treatment, the number of branches of the two lines was the same, but the plant height of the OE line was significantly higher than that of WT, and the plants grew more vigorously. After treatment with 150 mM NaCl, the plant heights of both lines decreased compared with the control, and the phenomenon of yellowing leaves appeared in both, but the number of branches of OE increased, indicating that OE plants were more salt-tolerant phenotypically. Under drought treatment, the WT plants were short, the plant height was significantly lower than that of the control, the number of branches decreased and the leaves showed a tendency to turn yellow; the plant height of the OE plants decreased slightly compared with the control, but the leaves were still green, indicating that the OE line was more drought-tolerant. Under ABA treatment, the plant heights of both lines increased compared with the control, but the plant height of OE was higher than that of WT, and the number of branches was more than that of WT, indicating that the promoting effect of ABA on plant growth was more obvious in OE( Figure 17 ).

[0219] 2.4.3.1 Effects of different treatments on the morphology of transgenic alfalfa

[0220] Under salt stress and drought stress, the growth of WT and OE plants was inhibited to varying degrees, while the application of ABA significantly promoted the growth of both WT and OE plants. Compared with CK, the plant heights of WT and OE lines under salt stress decreased by 21.55% and 18.88% respectively; the stem diameters decreased by 52.09% and 5.48% respectively; the root lengths showed a downward trend, and there was a significant difference in root length between the two lines (P < 0.05); the number of branches of WT showed a downward trend while that of OE showed an increasing trend. This indicates that compared with WT, the growth of OE lines was inhibited to a lesser extent by salt stress. Under drought stress, the plant heights of WT and OE lines decreased by 25.86% and 16.40% respectively compared with CK; the stem diameters decreased by 61.74% and 10.62% respectively; the root lengths and the number of branches were also affected by stress and showed a decreasing trend, and the drought tolerance of transgenic MsSNAT1 alfalfa was higher than that of the wild type. Foliar spraying of 100 μmol·L -1 of ABA promoted the growth of WT and OE lines to varying degrees. The plant heights and stem diameters of WT and OE lines increased by 70.69%, 31.91% and 20.58%, 30.14% respectively compared with CK; the root lengths also increased to varying degrees, which helped the plants absorb water and nutrients better, and the application of ABA promoted the branching of OE lines more. This shows that exogenous spraying of an appropriate amount of ABA can promote the growth of WT and OE alfalfa( Figure 18 ).

[0221] 2.4.3.2 Effects of different treatments on the biomass of transgenic alfalfa

[0222] As Figure 19 shown, under salt stress, the dry weights and fresh weights of WT and OE lines were both lower than those of the control, and there was a significant difference between the two lines. The fresh weight and dry weight of WT decreased significantly by 37.04% and 61.54% respectively compared with CK, while OE only decreased by 13.06% and 22%. Under drought stress, the fresh weight and dry weight of OE lines were 85.71% and 49.02% higher than those of WT respectively. Under ABA treatment, the biomass of both lines showed an increasing trend. Applying an appropriate amount of ABA helps improve the stress resistance of seedlings, promotes the rapid growth and development of roots, and increases the biomass accumulation of plants.

[0223] 2.4.3.3 Effects of different treatments on the chlorophyll of transgenic alfalfa

[0224] As Figure 20As shown, compared with the control, both salt treatment and drought treatment caused yellowing and withering of the leaves of WT and OE, a decrease in the relative chlorophyll content, and there were significant differences between the two lines (P < 0.05). Under salt stress, the chlorophyll content of WT decreased by 37.51% compared with the control, while that of OE decreased by 20.32%. Under drought stress, the chlorophyll content of WT decreased by 32.89% compared with the control, and that of OE decreased by 16.67%; while there was no significant difference in the chlorophyll content after applying ABA compared with the control. This indicates that the chlorophyll content of transgenic alfalfa is less inhibited by salt and drought stress, and applying ABA has no significant effect on the relative chlorophyll content of alfalfa.

[0225] 2.4.3.4 Effects of different treatments on the antioxidant capacity of transgenic alfalfa POD SOD CAT APX MDA

[0226] To further explore the mechanism of MsSNAT1 regulating the stress resistance of alfalfa, the antioxidant capacity of transgenic plants under different treatments was evaluated. The results showed ( Figure 21 ) that there were no significant differences in the measured indexes of each group of WT and OE under the water treatment. Compared with WT, the antioxidant enzyme activities (SOD, POD, CAT, APX) of the OE line under salt treatment were significantly higher by 40.78%, 43.43%, 48.66%, and 19.76%; the MDA content was significantly lower than that of WT (P < 0.05), indicating that the OE line had stronger antioxidant capacity. Under drought treatment, the antioxidant enzymes of the OE line were significantly higher than those of WT (P < 0.05), and the MDA content was 20.27% lower than that of WT, indicating that the degree of damage to the cell membrane of the OE line by drought stress was smaller. Under ABA treatment, the antioxidant enzyme activities of both lines increased, and the increasing trend was more obvious in the OE line. The MDA content had no significant difference from that of the control group. Therefore, overexpressing MsSNAT1 can enhance the antioxidant enzyme activity of alfalfa to scavenge reactive oxygen species, reduce the MDA content, protect cells from oxidative damage, and thus enhance stress resistance. In addition, ABA can also act together with melatonin to enhance the antioxidant capacity of plants.

[0227] 2.4.3.5 Effects of different treatments on the osmotic adjustment ability of transgenic alfalfa

[0228] As Figure 22As shown, compared with the control group, the proline content of WT and OE lines increased significantly under salt stress, and OE was significantly 73.62% higher than WT (P < 0.05); under drought stress, the proline content of WT and OE lines increased by 139% and 220% respectively; the application of ABA significantly accumulated proline in WT and OE lines. These results indicate that under salt and drought stress, the proline content of the OE line is significantly higher than that of WT, and it has a stronger ability to alleviate the osmotic stress of plants; the application of ABA can improve the osmotic adjustment ability of alfalfa, thereby increasing the proline content in the plant body.

[0229] 2.4.3.6 Effects of Different Treatments on the Expression of Antioxidant Enzyme Genes in Transgenic Alfalfa

[0230] The genes for antioxidant enzyme synthesis showed an increasing trend under different treatments. In order to resist and adapt to the damage caused by abiotic stress, plants initiate a stress response mechanism and activate the expression of antioxidant enzyme genes. Under normal culture conditions, there was no significant difference in the expression levels of antioxidant enzyme genes between the two lines. Under salt stress and drought stress, the expression levels of MsSOD, MsCatalase4, MsAPX, and MspxdC genes in the OE line were 52.06%, 67.12%, 34.09%, and 72.84% higher than those in WT, and 72.83%, 16.15%, 80.89%, and 12.24% respectively. This shows that under salt and drought stress treatments, alfalfa can initiate its own defense mechanism to resist stress, while the resistance of the wild type has a certain limit. The transgenic type can further increase the expression level of antioxidant enzyme genes, scavenge ROS, and enhance its own stress resistance. Under ABA treatment, the expression levels of antioxidant enzyme genes in both WT and OE lines showed an upward trend, indicating that 100 μmol ABA can promote plant growth ( Figure 23 ).

[0231] In summary, the experimental results show that the overexpression of the MsSNAT1 gene enhances the stress resistance of alfalfa. The overexpression of the MsSNAT1 gene promotes the increase in endogenous melatonin content and increases the expression of melatonin synthase genes under salt stress. Compared with the wild type, the transgenic lines showed a more significant increasing trend in antioxidant enzyme activity and osmotic adjustment substances under salt and drought stress, with a lower MDA content and less negative impact. In addition, the increase in endogenous melatonin content in the transgenic lines, together with exogenous ABA, enhances the antioxidant ability and osmotic adjustment ability of plants, and significantly improves the stress resistance of plants.

[0232] It is obvious to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, in all respects, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes that fall within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

Claims

1. The MsSNAT1 gene of alfalfa, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A transgenic vector containing the alfalfa MsSNAT1 gene according to claim 1.

3. A recombinant strain or recombinant cell containing the alfalfa MsSNAT1 gene according to claim 1.

4. Use of the alfalfa MsSNAT1 gene overexpression according to claim 1 in improving plant salt tolerance.

5. Use of the alfalfa MsSNAT1 gene according to claim 1 in improving the resistance quality of alfalfa and breeding of salt-tolerant alfalfa varieties.

Images