Application of MsERF07 gene in regulation and control of plant drought resistance

Abstract

The invention discloses application of an MsERF07 gene in regulation and control of drought resistance of plants, and is characterized in that a precursor sequence of the MsERF07 gene is as shown in SEQ ID No.1, and a mature sequence of the MsERF07 gene is as shown in SEQ ID No.2. The invention further discloses application of the MsERF07 gene in regulation and control of drought resistance of plants. The gene or an expression substance for regulating and controlling the gene or a substance for regulating and controlling the activity or content of protein can be applied to the aspect of regulating and controlling the drought resistance of plants, the drought resistance of the plants can be improved by over-expressing the MsERF07 gene, and the gene has a positive effect on accelerating and improving the variety of medicago sativa.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of MsERF07 gene in regulating plant drought resistance. Background Art

[0002] Alfalfa (Medicago sativa L.) is a perennial herb in the genus Alfalfa of the Leguminosae family. Most cultivated species are autotetraploid, cross-pollinated, and semi-self-incompatible [1]. The rhizome is well developed, and the plant height is 30 cm-100 cm. The leaves are pinnate-trifoliate; the stipules are large, ovate-lanceolate, and the petioles are shorter than the leaflets; the leaflets are long ovate, oblong ovate, or linear-ovate, equal in size, or the terminal leaflets are slightly larger, 1-4 cm long, with more than 1 / 3 of the margin serrated, the upper surface of the leaf is glabrous, the lower surface is covered with appressed soft hairs, and the lateral veins are 8-10 pairs; the terminal petiole is slightly longer than the lateral petioles. It prefers warm and semi-humid to semi-arid climates, is light-loving, and is a long-day plant. When the temperature is above 20°C and the soil pH is preferably pH 7, it grows slowly during the seedling stage.

[0003] AP2 / ERF transcription factors are important regulatory elements in plants, including five subfamilies: AP2 (APETALA2), ethylene response factor (ERF), drought response element binding protein (DREB), RAV and Soloist. They play an important role in plant response to environmental stress, growth and development regulation and signal transduction. NO, as an important gaseous signal molecule in plants, can mediate the expression of AP2 / ERF transcription factors, but how it functions in alfalfa drought resistance has not been reported. This study discovered the gene MsERF07 that strongly responds to drought stress and NO signals, constructed an overexpression vector of MsERF07 using homologous recombination technology, obtained transgenic plants by Agrobacterium-mediated method, and statistically analyzed the phenotypic characteristics of alfalfa under drought stress and NO treatment, antioxidant enzyme activity and other stress resistance indicators, and deeply explored the drought resistance of transgenic plants from the morphological, physiological and biochemical levels. Summary of the invention

[0004] In order to achieve the above object, the present invention designs the application of MsERF07 gene in regulating plant drought resistance.

[0005] In order to achieve the above technical effects, the present invention is implemented through the following technical scheme: Application of the MsERF07 gene in regulating plant drought resistance, characterized in that the nucleotide sequence of the MsERF07 is shown in SEQ ID No.1, and the amino acid sequence of the MsERF07 is shown in SEQ ID No.2.

[0006] SEQ ID No.1: ATGCCGAAGAAGCGTGCAGGGAGGAAGAAGTTCAGAGAAACACGTCACCCTGTGTACCGTGGTGTAAGAAAGAGGGATTCCGGGAAGTGGGTTTGTGAAGTAAGAGAACCTAACAAGAAAACCAGAATTTGGCTAGGAACATTTCCTACACCGGAGATGGCGGCGCGTGCACACGATGTTGCGGCGATTGCGTTGAGGGGAAGGTCTGCTTGTCTTAACTTTGCTGATTCTGCATGGAGGTTGCCGGTCCCGGCTACGGCGGATGCAAGGGATATTCAAAAGGCAGCGGCTGAAGCAGCGGAGGCTTTCAGGCCGGAGAGTGTGTTTGAGAATAGTGAGGAGAGGAAGGATAGTGAATCTTCATCAACGGTGGCGGTGGCAGAGACGGTGATGGAGCAACGAGAGGAAGAAGAGGATACAGTGCCGGAGTATTTGAGAAACATGGTGCTAATGTCACCGGCGCATTACTGGGGAAGTGATTGTGGTGTTGCTGACGTGGAATTTGATGAAACTGAAATTTCATTGTGGAGTTATTCATTTTAG;

[0007] SEQ ID No.2: MPKKRAGRKKFRETRHPVYRGVRKRDSGKWVCEVREPNKKTRIWLGTFPTPEMAARAHDVAAIALRGRSACLNFADSAWRLPVPATADARDIQKAAAEAAEAFRPESVFENSEERKDSESSSTVAVAETVMEQREEEEDTVPEYLRNMVLMSPAHYWGSDCGVADVEFDETEISLWSYSF;

[0008] Furthermore, when amplifying MsERF07, it includes MsERF07-F and MsERF07-R; among them, the nucleotide sequence of MsERF07-F is shown in SEQ ID No.3; the nucleotide sequence of MsERF07-R is shown in SEQ ID No.4.

[0009] SEQ ID No.3: 5'-CGGGGGACGAGCTCGGTACCATGCCGAAGAAGCGTGCA-3';

[0010] SEQ ID No.4: 3'-TGCTCACCATGTCGACCTAAAATGAATAACTCCACAATGAA-5'

[0011] Furthermore, overexpression of MsERF07 improves plant drought resistance.

[0012] Furthermore, a recombinant vector for overexpressing MsERF07 was constructed using homologous recombination technology, and the recombinant vector was transferred into plants to improve the drought resistance of the plants.

[0013] Furthermore, the method of using homologous recombination technology to construct a recombinant vector overexpressing MsERF07 specifically includes digesting the target fragment MsERF07 and the expression vector with KpnⅠ and SalⅠ at 37°C, and performing a homologous recombination reaction with a homologous recombinase after digestion to form a recombinant vector overexpressing MsERF07.

[0014] Furthermore, the overexpression vector is a pCAMBIA1300-35S-EGFP empty vector.

[0015] Furthermore, the MsERF07 gene improves the water retention capacity of plants and enhances their drought resistance by increasing the content of plant osmotic regulating substances and the activity of antioxidant enzymes to alleviate membrane oxidative damage.

[0016] Furthermore, the application is specifically: using Agrobacterium-mediated method to introduce MsERF07 gene into the plant whose drought resistance is to be improved to obtain transgenic plant.

[0017] Furthermore, the plant is selected as alfalfa.

[0018] The beneficial effects of the present invention are:

[0019] The invention discloses the application of MsERF07 gene in regulating plant drought resistance. The drought resistance of plants can be improved by overexpressing MsERF07, which has a positive effect on accelerating the improvement of alfalfa varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0021] Figure 1 This is a schematic diagram of the expression of MsAP2 / ERF under drought (PEG) and drought (PEG) + NO (SNP) stress detected by qRT-PCR of the present invention;

[0022] Figure 2 This is a PCR detection diagram of the alfalfa MsERF07 gene after cloning of the present invention;

[0023] Figure 3 The pCAMBIA1300-35S-EGFP plasmid of the present invention is verified by restriction enzyme cutting and PCR detection of bacterial solution;

[0024] Figure 4 This is a double enzyme digestion identification and Agrobacterium transformation PCR detection diagram of the present invention;

[0025] Figure 5 Schematic diagram of the expression of MsERF07 gene in various parts of alfalfa

[0026] Figure 6 This is a schematic diagram of tissue-specific expression of the MsERF07 gene of the present invention in alfalfa;

[0027] Figure 7 This is a diagram showing the acquisition of the MsERF07 transgenic plant of the present invention;

[0028] Figure 8 This is a PCR detection diagram of the alfalfa leaf DNA with overexpression of the MsERF07 gene of the present invention;

[0029] Fig. 9 This is a diagram showing the hardening and transplanting of the transgenic alfalfa plants of the present invention;

[0030] Fig.10 This is an analysis diagram of the expression level of the MsERF07 transgenic plant of the present invention;

[0031] Fig.11 The figure is a phenotypic analysis diagram of wild type and transgenic alfalfa under drought stress regulated by NO of the present invention;

[0032] Fig.12 The figure is an analysis diagram of the water loss rate and relative water content of the detached leaves of the transgenic plant of the present invention;

[0033] Fig.13 This is the effect of exogenous NO on photosynthetic pigments of transgenic plants under drought stress.

[0034] Fig.14 This is a diagram showing the effect of exogenous NO on the antioxidant enzyme activity (AC) and osmotic regulation system (DF) of transgenic plants under drought stress;

[0035] Fig.15 The effect of exogenous NO on the H2O2 (A) and O2.- (B) content analysis of transgenic plants under drought stress;

[0036] Fig.16 The figure shows the effect of exogenous NO on membrane lipid antioxidant capacity (A) and MsERF07 gene expression (B) of transgenic plants under drought stress. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Example 1

[0039] The technical problem to be solved in this embodiment is how the MsERF07 gene improves the drought resistance of alfalfa, and the following steps are adopted:

[0040] 1) Identify and clone MsERF07 in alfalfa and perform bioinformatics analysis on it;

[0041] 2) Construction of overexpression vector of MsERF07 gene in alfalfa and genetic transformation, and analysis of drought resistance;

[0042] 3) Overexpress MsERF07 in alfalfa using artificial miRNA technology and analyze drought resistance;

[0043] I. Identification and bioinformatics analysis of the MsERF07 gene in alfalfa

[0044] 1. Methods:

[0045] 1.1 Identification of MsERF07 gene:

[0046] The 13 MsAP2 / ERF genes identified were further confirmed using the online tool SMART. The results showed that all 13 family members contained protein domains. Then, the expression levels of the 13 MsAP2 / ERF genes under drought stress were analyzed using qRT-PCR technology, and the most significant MsERF07 gene was screened out as a candidate gene. Figure 1 shown.

[0047] like Figure 2 As shown, the cDNA of the leaves of the alfalfa variety "Suntory" was used as a template, PCR amplification was performed with subcellular localization primers, and the size of the target fragment was detected by 1% agarose gel electrophoresis; the results showed that the size of the target fragment band was about 552bp, which was consistent with the expected target fragment size;

[0048] 2. Construction of the overexpression vector of MsERF07 gene in alfalfa and genetic transformation

[0049] 1. Methods:

[0050] 1.1 Construction of alfalfa MsERF07 gene overexpression vector

[0051] 1.1.1 Primer design and synthesis

[0052] The sorted CDS sequence of the alfalfa MsERF07 gene was submitted to the Takare Bio official website (https: / / www.takarabio.com / learning-centers / cloning / primer-design-and-other-tools) to design specific primers.

[0053] The forward primer MsERF07-F is:

[0054] The reverse primer MsERF07-R is: (The italic parts are the restriction sites of KpnⅠ and SalⅠ, and the underlined parts are the homologous sequences), synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0055] 1.1.3 Amplification of target fragment of MsERF07 gene

[0056] The obtained cDNA was used as an amplification template, and a recombinant vector for subcellular localization was constructed using homologous recombination. The reaction system is shown in Table 1.

[0057]

[0058] Table 1 Target fragment amplification system

[0059] The above reaction system was added to a centrifuge tube and mixed (placed on ice), further mixed using a low-speed centrifuge, and the target fragment was amplified in a PCR instrument.

[0060] The reaction conditions for amplification are shown in Table 2:

[0061]

[0062]

[0063] Table 2

[0064] The obtained PCR products were first subjected to 1% agarose gel electrophoresis, and then the agarose gel containing the target band was cut out using a sterile gel cutter. The amplified products were recovered and purified using a gel recovery kit, and the recovered and purified products were stored in a -20°C refrigerator for subsequent use.

[0065] 1.1.3 Expression vector linearization

[0066] Take out the empty plasmid from the -80℃ ultra-low temperature freezer and quickly place it on ice. Use a pipette to accurately draw 500μL of bacterial solution and inoculate it into 50mL LB liquid culture medium containing Kan antibiotics. Place the culture medium in a 37℃ constant temperature shaker and culture it overnight at 260r / min for about 12h. Use a spectrophotometer to measure the bacterial solution OD 600 =0.8, extract the plasmid from the pCAMBIA1300-35S-EGFP empty vector according to the steps in Appendix 3, and finally perform double digestion of the extracted empty plasmid vector with restriction endonucleases Kpn I and SalI to produce sticky ends. The reaction system is shown in Table 3

[0067]

[0068] Table 3 Double enzyme digestion system

[0069] After adding the sample to the above system, centrifuge it in a low-speed centrifuge, then place it in a PCR instrument and digest it at 37℃ for 30min, and heat it at 85℃ for 5s to inactivate the enzyme. Use 1% agarose gel for electrophoresis to determine the desired target band, and recover the target band according to the steps in Appendix 2. Finally, store the recovered product in a -20℃ refrigerator.

[0070] 1.1.4 Homologous recombination reaction

[0071] The MsERF07 gene amplification product obtained in the above steps and the restriction product of the pCAMBIA1300-35S-EGFP empty vector were subjected to homologous recombination reaction using homologous recombinase. The reaction system is shown in Table 4.

[0072]

[0073] Table 4 Homologous recombination reaction system

[0074] 1.1.5 Transformation of recombinant expression vector into Escherichia coli

[0075] Take out the prepared E. coli competent cells DH5α from the -80℃ refrigerator, place them on ice to melt until they are in an ice-water mixed state, add 10μL of the above-mentioned ligation product to the center of 100μL DH5α cells, and mix them with a pipette tip; after an ice bath for 30 minutes, place them in a 42℃ water bath for 45 seconds, and immediately transfer the centrifuge tube to ice for 2 minutes to avoid shaking the centrifuge tube; add 750μL of LB liquid culture medium without antibiotics to the centrifuge tube, place the centrifuge tube in a 37℃ environment for 5 minutes, and then perform constant temperature shaking activation culture at 37℃ at a speed of 260r / min for about 1 hour; aspirate 100μL of the transformed bacteria and spread them on LB (containing 50μg / mL Kan) solid culture medium, and culture them upside down at 37℃ overnight.

[0076] 1.1.6 PCR verification of bacterial culture and restriction enzyme digestion identification of recombinant plasmid pCAM-MsERF07-EGFP

[0077] After 12 hours of cultivation, single colonies can be seen on the LB solid culture medium. Use a gun tip to pick up single colonies and inoculate them into 5 mL LB liquid culture medium containing 50 μg / mL Kan antibiotics. Place the culture medium in a 37°C constant temperature shaker and shake at 210 rpm for about 10 hours. Use bacterial liquid PCR for identification, and refer to Table 3-2 in 1.1.3 for its system. Verify that it is a positive clone bacterial liquid and send it to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. Then extract the plasmids of these bacterial liquids and perform enzyme digestion verification according to the enzyme digestion system and conditions in Table 1 in 1.1.3. The appropriate plasmid after enzyme digestion verification is the subcellular expression vector, named pCAM-MsERF07-EGFP.

[0078] 1.1.7 Transformation of Agrobacterium with the recombinant expression vector pCAM-MsERF07-EGFP

[0079] The constructed pCAM-MsERF07-EGFP plasmid was transformed into Agrobacterium competent GV3101. The main transformation steps were as follows: two tubes of frozen Agrobacterium competent cells were taken from a -80°C refrigerator, quickly placed on ice, and 10 μL of pCAM-MsERF07-EGFP plasmid and pCEGFP plasmid were added to the center of the bacterial solution respectively; then LB liquid culture medium without antibiotics was added to the bacterial solution; the cultured bacterial solution was centrifuged at 6000 r / min for 1 min, part of the supernatant was discarded, and about 100 μL of the bacterial solution was gently blown and resuspended with a pipette tip, and incubated at 28°C for 48 h; a single colony with smooth edges was picked up with a pipette tip, 10 mL of LB liquid culture medium (containing Kan and Rif) was added to a 50 mL centrifuge tube, and incubated in a shaking incubator for 12-18 h, and the cultured bacterial solution was used as a template for colony PCR identification, and the bacterial solution with the target band was frozen at -80°C for later use.

[0080] 1.2 Cloning of MsERF07 gene

[0081] Using the cDNA of the leaves of the alfalfa variety "Suntory" as a template, PCR amplification was performed using subcellular localization primers, and the size of the target fragment was detected by 1% agarose gel electrophoresis. The results showed that the size of the target fragment band was about 552bp, which was consistent with the expected target fragment size ( Figure 3 (as shown in Figure A).

[0082] 1.1.7 Bacterial liquid PCR verification

[0083] As shown Figure 3 As shown in Figure B, a band with the same size as the target fragment was amplified, indicating that homologous recombination was successful and the recombinant product was transformed into Escherichia coli. The correct cloning vector was named MsERF07-pEGFP and used in subsequent subcellular localization experiments.

[0084] 1.1.8 Enzyme digestion identification of pEGFP-MsERF07 vector

[0085] The recombinant plasmid pCAM-MsERF07-EGFP was digested with Kpn I and Sal I and the results showed that the small fragment had a band around 552 bp ( Figure 3 The expected result of the target gene was met, and its transient expression vector plasmid was named pCAM-MsERF07-EGFP.

[0086] 1.3 Subcellular localization vector transformation into Agrobacterium

[0087] pCAM-MsERF07-EGFP was transformed into Agrobacterium competent cells, and its target band was detected by PCR, such as Figure 4 As shown in Figure B, bacterial cultures No. 1-4 amplified target bands consistent with expectations, indicating that the alfalfa pCAM-MsERF07-EGFP subcellular localization vector was successfully transformed into Agrobacterium.

[0088] 1.3.1 Fluorescence detection of pEGFP-MsERF07 fusion protein

[0089] The protein subcellular localization was predicted by BUSCA, and the MsERF07 protein was localized in the cell nucleus. In order to further reveal the location and distribution of the gene in various organelles in alfalfa cells, we transformed the successfully constructed pCAM-MsERF07-EGFP into tobacco leaves. After culturing in a dark incubator for 12 hours, the tobacco was transferred to a light station incubator for 48 hours. Finally, the transformed tobacco was observed with a laser confocal microscope to capture the laser signal. The results showed that the MsERF07-EGFP fusion protein was not only strongly expressed in the cell nucleus, but also in the cell membrane, indicating that the protein has multiple functions or plays different roles in different parts of the cell. The results are as follows: Figure 5 shown.

[0090] Figure 5 pCEGFP: blank control; MsERF07-pCEGFP: MsERF07-pCEGFP fusion protein; red, blue, yellow, and green arrows represent cell membrane, nucleus, cytoplasm, and chloroplast, respectively.

[0091] 1.3.2 Differential expression of MsERF07 gene in different tissues

[0092] qRT-PCR analysis of the tissue-specific expression of the MsERF07 gene in the alfalfa variety "Suntory" showed that in the alfalfa variety "Suntory", the expression level of the MsERF07 gene was the lowest in the flower, the expression levels in the seeds and stems were not significantly different, the expression level in the root was the highest, and the expression level in the leaf was the second highest. In the alfalfa variety "Suntory", there were significant differences in the expression of MsERF07 in different tissues, among which the expression level in the root was the highest. The results are shown in Figure 2. Figure 6 shown.

[0093] Figure 6 The one with the lowest expression level was used as the control; different letters indicate significant differences among treatments (P<0.05).

[0094] 1.4 Agrobacterium-mediated genetic transformation of alfalfa and production of transgenic plants

[0095] 1.4.1 Cultivation of sterile alfalfa seedlings

[0096] Preparation: Prepare 1 / 2MS solid culture medium and sterilize it in an autoclave, take it out and wait for it to cool and solidify for later use. Select alfalfa seeds with full and uniform grains, soak them in running water for 40 minutes, stir them and discard the small amount of seeds floating on the water surface, and put the seeds that sink to the bottom of the water on the clean bench for later use.

[0097] Sterilization: Place the seeds in 75% ethanol for disinfection for 45 seconds, then rinse the seeds with sterile water five times, use 0.1% HgCl2 solution to further disinfect the seeds for 5 minutes, and then rinse them with sterile water five times. Then soak them in sterile water for 30 minutes, pour out the water, and use sterile filter paper to absorb the residual water on the surface of the seeds.

[0098] Sowing: Carefully pick up the seeds with sterilized tweezers and sow them evenly in 1 / 2MS solid culture medium (culture conditions: temperature 25°C, photoperiod 16h light / 8h dark, light intensity 4000LX, culture time 7d).

[0099] 1.4.2 Agrobacterium-mediated genetic transformation of alfalfa

[0100] How to do it:

[0101] ① Prepare bacterial solution: Take out the bacterial solution containing the target band from the -80℃ refrigerator, add 50mL LB liquid medium (containing 50μg / mL Kan and 50μg / mL Rif), place it in a shaker at 28℃ and 210rpm, and shake until OD 600 =0.6. Finally, the bacterial solution was resuspended in MS liquid medium (containing 0.1mmol / L AS) and the bacterial solution concentration OD 600 =0.6 is appropriate.

[0102] ② Cut seedlings: Cut the hypocotyl (about 1 cm) of 7-day sterile seedlings and place them in sterile water first.

[0103] ③Infect hypocotyls: first pour out the sterile water carefully, then pour the MS resuspended infection solution into the hypocotyls to cover them. After 10 minutes of infection, pour out the infection solution and use sterile dry filter paper to absorb the residual bacterial solution on the surface of the hypocotyls.

[0104] ④ Inoculation: Inoculate into modified SH solid culture medium and co-cultivate for 3 days (dark incubator).

[0105] ⑤ Use modified SH liquid medium containing 400 mg / L Carb to sterilize the material, transfer it to callus medium, and carry out callus culture for 20 days. During this period, subculture is carried out every 10 days. Select callus tissues with compact and moist appearance, uniform size and good growth, and transfer them to MSO differentiation medium containing 50 μg / mL Kan or 15 mg / L Hyg for screening culture to promote bud differentiation. During the differentiation culture process, subculture is carried out every 20 days, and the Carb concentration is reduced by 1 / 2 each time.

[0106] ⑥ After about 60 days of culture, the hypocotyls grow adventitious buds and are transferred to rooting medium (1 / 2MS + 0.5mg / L NAA) for rooting. After rooting, they are temporarily identified as transformed seedlings and are preserved for later propagation.

[0107] 1.5 Identification of positive plants

[0108] Take 0.5 g of leaves from sterile alfalfa seedlings to extract DNA for PCR detection (see Appendix 5 for specific steps), use the DNA of wild-type (WT) alfalfa as the negative control, the Agrobacterium solution as the positive control, and water as the blank control.

[0109] To detect the kanamycin resistance marker gene NPTⅡ, specific PCR primers NP TⅡ-F: GCTATGACTGGGACAACAG, NPTⅡ-R: ATACCGTAAAGCACGAGGAA were used. The primers were designed for the PCR product of the transgenic positive plants. The length of the target fragment was 676bp through PCR amplification. Verify whether the kanamycin resistance marker gene NPTⅡ was successfully integrated into the transgenic plants. The reaction system is as follows: 10μL 2×Taq PCRMaster Mix, 1μL NPTⅡ-F, 1μL NPTⅡ-R, 1μL DNA, 6.5μL ddH2O. PCR amplification conditions: 94℃ pre-denaturation for 2min; 94℃ 45s; 60℃ 45s, 72℃ 20s, 35 cycles; 72℃ extension for 10min; 4℃ storage.

[0110] 1.4.4 Hardening and transplanting of positive plants

[0111] Preparation before hardening the seedlings: sterile water, tweezers and culture dishes sterilized in an autoclave.

[0112] Seedling hardening process: After the transgenic seedlings in the culture dish are moved to 1 / 2 of the culture medium and the root system is well developed and the above-ground part is on top of the bottle cap, loosen the bottle cap and place it in a light incubator for 12 hours. Add 50 mL of sterile water and gently place the bottle cap on the bottle body for 3 days before removing it. Wait for about 5 days to allow the sterile seedlings to gradually adapt to the external environment.

[0113] Transplantation process: Use tweezers to cut the bottom culture medium to avoid the root system, carefully remove the transgenic plants from the culture bottle, rinse the residual solid culture medium on the roots with sterile water to reduce the contamination of the transplanting matrix, transfer to the nutrient solution for hydroponics for about 20 days, and move to a small flowerpot (15 cm in diameter) for room temperature culture (nutrient soil: vermiculite = 3:1).

[0114] Management after transplanting: In the early stage after transplanting, keep the soil humidity high to promote the recovery of root growth. Gradually reduce the soil humidity in the later stage to prevent root rot. Check the sterile seedlings after transplanting every day to detect and prevent diseases and insect pests in time to ensure their healthy growth.

[0115] 1.4.5 qRT-PCR analysis of transgenic plants

[0116] RNA of transgenic plants that were positive by PCR was extracted, and the relative expression level of MsERF07 gene was detected by qRT-PCR. qRT-PCR primers were designed according to the target gene, and β-Actin was used as the internal reference gene.

[0117] 1.5 Resistance analysis of transgenic plants

[0118] 1.5.1 Experimental treatment

[0119] After the plants were cultured at room temperature for 30 days, wild-type (WT) and transgenic (OE) alfalfa strains with consistent growth were selected for treatment. The control group (T1) was watered normally, and the treatment groups were: T2: 10% PEG-6000; T3: 0.1mmol / L SNP + 10% PEG-6000. The T2 treatment group was irrigated with 10% PEG-6000 once a day for 7 days; the T3 treatment group was pretreated with 0.1mmol / L SNP 3 days in advance, sprayed until the leaves were about to drip, and treated once every 24 hours. The SNP used must be freshly prepared and used. Sampling was carried out 7 days after treatment, and 3 biological replicates were set for each physiological indicator. The samples were stored in a -80℃ refrigerator for future use.

[0120] 1.5.2 Determination of growth characteristics

[0121] Drought treatment and phenotypic observation of transgenic plants:

[0122] Detached leaf water loss rate:

[0123] Preparation: Select leaves with similar growth conditions and at the same growth stage (middle and upper leaves, 3rd-year leaves) as experimental materials, scissors, tweezers, culture dishes, electronic balance, etc.

[0124] Leaf treatment and weighing: Use tweezers to pick mature leaves of wild-type and transgenic plants, rinse the surface and then use filter paper to absorb the moisture on the leaf surface.

[0125] Observation and weighing of water loss process: Leaves were placed under conditions similar to the plant growth environment to simulate the natural water loss process, and the leaves were weighed at predetermined time points.

[0126] Relative Water Content (RWC):

[0127] Preparation: Select healthy plant leaves as the measurement object and quickly cut the required amount of leaf tissue with scissors.

[0128] Fresh weight determination: The fresh weight (W1) of the cut plant material was weighed on a scale and placed in a 50 mL centrifuge tube. Sterile water was then added to the centrifuge tube to soak the leaves for 24 h, and the leaves were taken out and the surface moisture was wiped dry to obtain the saturated fresh weight (W2).

[0129] Dry weight determination: Open the oven in advance and raise the temperature to 105°C to ensure that the leaves in the aluminum box can quickly reach a constant weight during the drying process. Finally, place them in a desiccator and cool them to room temperature before weighing them (W3).

[0130] Formula for calculating RWC: RWC = (W1-W3 / W2-W3)*100

[0131] 1.5.3 Determination of photosynthetic system

[0132] Chloroplast pigment content: refer to the chlorophyll meter living leaf measurement method and organic solvent extraction colorimetric method.

[0133] Pigment extraction: Weigh 0.5g of fresh alfalfa leaves, wipe the surface of the tissue clean, put it in a mortar, add a small amount of quartz sand and 2-3mL 95% ethanol, grind into a homogenate. Add 10mL of ethanol, continue grinding until the tissue turns white, and let it stand in the dark for 10 minutes (under weak light).

[0134] Filtration: Filter the ground homogenate into a 50mL centrifuge tube, add a small amount of ethanol solution to wash the mortar several times, filter the washing solution into a centrifuge tube, and dilute to the same volume. Determine absorbance: Pour the pigment extract into a quartz cuvette, use 95% ethanol as a blank control, and measure the absorbance at specific wavelengths (665nm, 649nm and 470nm) (under weak light).

[0135] Calculation: The absorbance and the absorption coefficient at the corresponding wavelength are calculated using the corresponding formula.

[0136] 1.5.4 Determination of antioxidant system

[0137] Antioxidant enzyme activity:

[0138] Preparation of enzyme solution: Weigh 0.5g sample into a pre-cooled mortar, add 2ml pH=7.8 phosphate buffer, grind in an ice bath, pour the homogenate into a centrifuge tube and then add 3ml buffer for washing, place at 4℃, 5000r / min centrifuge for 10min, pour the supernatant into a test tube (enzyme solution), and store at 0-4℃ for later use.

[0139] Enzyme activity determination: SOD, POD, and CAT were determined according to the methods of Huang et al., Shi et al., and Aebi, respectively.

[0140] 1.5.5 Determination of osmotic regulating substances

[0141] Proline (Pro) content: Sample preparation, weigh 0.5g of alfalfa leaves with different treatments and place them in a test tube, add sulfosalicylic acid solution, extract in a boiling water bath for 10 minutes, cool and filter to obtain the filtrate as the proline extract. Proline determination: blank control (toluene solution), sample (proline red toluene solution with different treatments).

[0142] Plant cell membrane permeability: Sample preparation: Select alfalfa leaves and wash them with sterile water, then use filter paper to absorb the moisture on the leaf surface, and use a hole puncher with a diameter of 1 to drill small discs. Sample processing: Use a vacuum pump to evacuate the air in the cell gap for 20 to 30 minutes, place it in a water bath and boil it for 10 to 15 minutes, and then measure it with a conductivity meter after cooling.

[0143] Soluble protein content: Sample extraction: Take 0.5 fresh alfalfa sample, grind it into a homogenate with buffer solution, centrifuge it and take the supernatant for later use. Sample determination: Take an appropriate amount of sample extract, add Coomassie brilliant blue reagent and measure the absorbance.

[0144] 1.5.6 Membrane lipid oxidation system

[0145] Malondialdehyde (MDA) content: Sample preparation, weigh 0.5g of fresh alfalfa leaves, put them in a mortar, add a little quartz sand and 2mL 10% trichloroacetic acid, grind them into a homogenate, add 3mL 10% trichloroacetic acid for further grinding, and centrifuge. The supernatant obtained is the MDA extract. For sample determination, 2mL 10% trichloroacetic acid is used as a control, and the absorbance values ​​at 532nm and 600nm are determined by a spectrophotometer.

[0146] H2O2 and Content: Nitrotetrazolium chloride (NBT) staining and 3,3-diaminobenzidine (DAB) staining

[0147] 1.6 Results Analysis

[0148] 1.6.1 Cloning of MsERF07 gene

[0149] Using cDNA from the leaves of alfalfa variety "Suntory" as a template, PCR amplification was performed using homologous recombination primers, and then 1% agarose gel electrophoresis was used to detect the target fragment. Figure 4 As shown in A, the size of the target fragment is about 552 bp, which is consistent with the expected size of the target fragment.

[0150] 1.6.2 Bacterial liquid PCR verification

[0151] like Figure 4As shown in B, after the homologous recombination products were transformed into competent E. coli, bands of about 552 bp were amplified, which was consistent with the size of the target fragment, indicating that the homologous recombination was successful.

[0152] Figure 4 Figure A in the middle is the PCR product of MsERF07 gene of alfalfa; Figure B is the PCR of bacterial solution connected with vector pCAMBIA1300-MsERF07; where M: DNA Maker DL 2000; A.1-3: target fragment; B.1-3: monoclonal bacterial solution amplification PCR.

[0153] 1.6.3 Double enzyme digestion verification

[0154] like Figure 4 As shown in A, the sizes of the vector fragment and the target gene fragment were consistent with expectations, indicating that the alfalfa overexpression vector pCAMBIA1300-MsERF07 was successfully constructed.

[0155] 1.6.4 Agrobacterium transformation of overexpression vector pCAMBIA1300-MsERF07

[0156] The above recombinant vector pCAMBIA1300-MsERF07 was transformed into Agrobacterium competent cell GV3101. Figure 4 As shown in B, the bacterial suspension amplified bands with the same size as the target fragment, indicating that the pCAMBIA1300-MsERF07 overexpression vector of alfalfa was successfully transformed into Agrobacterium.

[0157] Figure 4 A is the double enzyme digestion identification of pCAMBIA1300-MsERF07 plasmid; B is the PCR product of Agrobacterium transformation; M: DNA Maker; A.1: double enzyme digestion product; B.1-4: transformation products.

[0158] 1.6.5 Agrobacterium-mediated genetic transformation of alfalfa

[0159] The genetic transformation process of alfalfa Figure 7 As shown, 7-day-old sterile seedlings ( Figure 7 First, cut the hypocotyl of 7-day-old alfalfa "Suntory" ( Figure 7 As shown in B), the transformed alfalfa pCAMBIA1300-MsERF07 overexpression vector Agrobacterium was used for infection and cultured in the dark for 3 days. The hormone ratio of the culture medium was: 2,4-D+6-BA+Carb (100 μL). The seedlings were washed and the Carb was reduced to 75 μL callus culture medium for 15 days to form callus tissue ( Figure 7 C), and on the 50th day, differentiation medium was used to screen for differentiation buds ( Figure 7D), and transferred to rooting medium on the 70th day for rooting ( Figure 7 E), and then transformed seedlings ( Figure 7 (as shown in F).

[0160] 1.7 Positive plant detection

[0161] 1.7.1 PCR detection of alfalfa overexpressing plants

[0162] Wild-type alfalfa DNA and ddH2O were used as negative and blank controls, and pCAMBIA1300-MsERF07 overexpression vector Agrobacterium culture was used as a positive control. Figure 8 PCR detection of leaf DNA (as shown in Figure A) showed that the expression vector contained the NPT II resistance selection marker gene (676 bp). Figure 7 As shown in Figure B, transgenic alfalfa strains 1-7 were all identified as positive, indicating that the MsERF07 gene was successfully overexpressed in alfalfa.

[0163] Figure 7 M: DNA Maker DL 2000; C: wild-type plants; +: positive control; 1-11: transgenic plants

[0164] 1.7.2 Hardening and transplanting

[0165] like Fig. 9 As shown in Figure A, when the transgenic and wild-type alfalfa seedlings grow to the bottle mouth, they can be hardened. Loosen the bottle cap and add an appropriate amount of sterile water. After 2 to 3 days, completely open the bottle cap. After 7 days, pull it out of the culture medium with the roots, wash the roots, and move it into the nutrient solution for culture ( Fig. 9 After culturing in the nutrient solution for 20 days, the plants were transplanted into small flower pots with a nutrient soil: vermiculite ratio of 3:1. The alfalfa plants grew well with lush roots, and the transplant survival rate was 100% ( Fig. 9 (as shown in Figure C).

[0166] 1.7.3 Analysis of expression levels in transgenic plants

[0167] Five transgenic plants were randomly selected from the identified positive plants to extract RNA, and the expression levels of wild type and transgenic were detected by qRT-PCR. Fig.10 As shown, among the overexpression lines, OE1 and OE4 had the highest expression levels, which were 10.6 and 12.8 times higher than those of the wild-type line, respectively. The expression levels of the remaining three lines were also higher than that of the wild-type (7.4, 8.2, and 5 times higher), indicating that the MsERF07 gene was successfully highly expressed in the plant.

[0168] 1.8 Analysis of drought resistance of overexpressed MsERF07 gene

[0169] 1.8.1 Analysis of growth characteristics of transgenic plants

[0170] 1.8.1.1 Phenotype

[0171] like Fig.11 As shown in the figure, under normal growth conditions, the phenotypes of the overexpression (OE) strains and the wild-type strains (WT) did not show significant differences. However, when the plants were subjected to 7 days of drought stress, the wilting degree of the OE strains was significantly lighter than that of the WT strains. Subsequently, they were treated with SNP, which is an exogenous substance designed to induce stronger drought resistance in plants. The results showed that the drought resistance of both the OE and WT strains was significantly improved. However, it is worth noting that in the drought stress environment regulated by exogenous nitric oxide (NO), although the drought resistance of both was enhanced, the top of the WT plant showed more obvious signs of wilting than the OE strain. This shows that although the SNP treatment generally improves drought resistance, the OE strain shows a superior performance in coping with this specific environmental stress.

[0172] 1.8.1.2 Analysis of water loss rate and relative water content of detached leaves

[0173] The water loss in plants is mainly through leaves. This characteristic makes the water loss state of detached leaves an important indicator for evaluating the drought resistance of plants. Therefore, by accurately measuring the water loss rate of plant leaves, a preliminary and effective analysis of the drought resistance of plants can be carried out. Fig.12 As shown in Figure B, as time went by, the water loss rate of detached alfalfa leaves showed an overall increasing trend. Along with this change, the leaves gradually showed a wilting state. Within 30 minutes after the leaves were detached, the water loss rates of both the wild type (WT) and the overexpression strain (OE) remained at a relatively close level, and no significant difference was observed between the two. However, when the time was extended to 60 minutes, the water loss rate of the detached leaves of the OE strain began to gradually decrease from that of the wild type. After 150 minutes of detachment, the water loss rate of the leaves of the WT strain was 42% higher than that of the OE1 strain, 36.6% higher than that of the OE2 strain, and 51.6% higher than that of the OE3 strain. The leaf phenotype also further showed that the WT showed a more wilting state compared with the OE (such as Fig.12 This change indicates that the MsERF07-OE strain has a stronger ability to retain water. Fig.12As shown in Figure C, under normal growth conditions, the relative water content of leaves of OE plants was higher than that of WT, and the relative water content of OE plants was 11.2%, 13.6% and 14.5% higher than that of WT, respectively. This indicates that the water content of transgenic alfalfa increased due to the up-regulation of the expression of the MsERF07 gene, thereby improving its drought tolerance.

[0174] 1.8.2 Photosynthetic Pigments Analysis

[0175] Chlorophyll is the core pigment of photosynthesis, which drives photosynthesis by absorbing light energy. Plants that are more drought-resistant will adapt to drought environments by reducing chlorophyll consumption and adjusting the rate of photosynthesis. Fig.13 As shown in the figure, after drought stress, the chlorophyll content of the OE strain was significantly higher than that of the WT strain (24.7%). After exogenous spraying of NO under drought stress, the chlorophyll content of both WT and OE strains increased significantly compared with the control, 13.1% and 7.6% higher than the control group, respectively, indicating that NO can increase the chlorophyll content of alfalfa and make it more drought-resistant. The chlorophyll a content showed that the content of the OE strain was significantly higher than that of the WT strain (25.9%) under the support of exogenous NO, and the chlorophyll b content showed that the content of the WT strain was lower than that of the OE strain, 30.3%, 29.4% and 31.6% lower, respectively. The role of carotenoids in photosynthesis is to absorb light energy and protect plants from photooxidative damage. When exogenous spraying of NO was applied, the carotenoid content of the OE strain was 11.9% higher than that of the WT strain.

[0176] 1.8.3 Antioxidant system analysis

[0177] 1.8.3.1 Analysis of antioxidant enzyme activity

[0178] like Fig.14 Figure A shows that under normal growth conditions, the SOD activity of the OE strain and the WT strain was not significantly different. However, when the plants were subjected to drought stress, the SOD activity of the OE strain increased significantly, 33.9% higher than that of the WT strain. When NO was sprayed exogenously, the SOD activity of both the OE strain and the WT strain increased significantly compared with the control group, increasing by 40.2% and 44.5%, respectively. It is worth noting that the SOD activity of the OE strain still maintained its advantage over the WT strain, which was 19.1% higher.

[0179] like Fig.14 The changes in POD activity are shown in Figure B. Under normal growth conditions, the POD activity of the OE strain is already higher than that of the WT strain, specifically 41.4% higher. When the plants are under drought stress mediated by exogenous NO, the POD enzyme activity of the OE strain is still higher than that of the WT strain (31.6% higher).

[0180] like Fig.14 Figure C shows the changes in CAT enzyme activity. Under normal growth conditions, the CAT enzyme activity of the OE strain was significantly higher than that of the WT strain, 57.1% higher. When the plants were subjected to drought stress, the CAT enzyme activity of both the OE and WT strains decreased. After exogenous spraying of NO, the CAT enzyme activity of both was restored and improved. Among them, the CAT enzyme activity of the OE strain increased by 10.1%, while that of the WT strain increased by 43.7%, making the enzyme activity of both close to the level of normal growth.

[0181] 1.8.4 Analysis of osmotic regulation ability

[0182] like Fig.14 As shown in Figure D, under normal growth conditions, the proline content of the WT strain is lower than that of the OE strain, while under drought stress, there is no significant difference between the WT and OE strains. After exogenous spraying of NO, the proline content of the OE strain is significantly higher than that of the WT strain. Under different treatments, the proline content of the OE strain is 51.8%, 1.9%, and 47.3% higher than that of the WT strain, respectively. Under drought conditions, plant cell membranes are often damaged, which can lead to the loss of electrolytes inside the cells, thereby increasing conductivity.

[0183] like Fig.14 As shown in Figure E, under drought stress, the relative conductivity of the WT and OE strains increased. After exogenous NO spraying, the relative conductivity decreased. Under different treatments, the relative conductivity of the OE strain was lower than that of the WT strain, 14.7%, 24.8%, and 51.7% lower, respectively. This proves that the MsERF07 gene reduces the degree of damage to the plant cell membrane, and the presence of exogenous NO makes the overexpression of MsERF07 more significant, making the plant more drought-resistant. In addition, soluble proteins can provide cell protection under drought conditions and alleviate metabolic disorders caused by water deficiency.

[0184] like Fig.14 As shown in Figure F, under normal growth conditions, the soluble protein content of the OE strain is slightly higher than that of the WT strain (1.8%), and when drought stress occurs, the content of the OE strain is significantly higher than that of the WT strain (7.9%). Under the support of exogenous NO, the soluble protein content of the OE strain is significantly higher than that of the WT strain (6.8%).

[0185] 1.8.5 Analysis of membrane lipid peroxidation

[0186] Hydrogen peroxide acts as a signal molecule in plants to regulate drought resistance. It can activate genes related to plant stress resistance and promote plant adaptation to environmental stress. Fig.15As shown in Figure A, the hydrogen peroxide content in the untreated OE strain and the WT strain was not significant. Under drought stress, the WT strain was slightly darker in color than the OE strain and had obvious mottled leaves, while the leaves treated with exogenous NO showed a light color, among which the leaves of the OE strain were smooth and lighter in color, indicating that under the regulation of NO, the expression level of the MsERF07 gene was further increased to reduce the oxidative damage of the plant.

[0187] like Fig.15 As shown in Figure B, under normal growth conditions, the color depth of the WT and OE strains was not significant, but after drought stress, the color of the WT strain was darker than that of the OE strain, indicating that more O2.- was accumulated in the WT strain and the degree of damage was greater than that of the OE strain. After spraying exogenous NO, the leaf color was significantly lighter than that of the drought-stressed strain, indicating that NO can help alfalfa leaves alleviate drought stress. An increase in MDA levels is generally regarded as a sign that plants or cells are experiencing oxidative stress or membrane lipid peroxidation. As shown in the figure, after simulated drought stress, the MDA content of the WT strain was significantly increased compared with the OE strain, while the MDA content after spraying exogenous NO was significantly lower than that of drought stress. Under different treatments, the OE strain showed stronger resistance to oxidative damage. Under different treatments, the MDA content of the WT strain was 45.8%, 37.2%, and 42.7% higher than that of the OE strain, respectively, indicating that the degree of damage suffered by the WT strain under different treatments was greater than that of the OE strain. Fig.16 As shown in Figure A.

[0188] In summary, this study showed that after overexpressing the MsERF07 gene, its proline content and soluble protein were significantly higher than those of the wild type, while its relative conductivity was lower than that of the WT strain. The content of membrane lipid oxidation product MDA showed that the MsERF07-OE strain was significantly lower than that of the WT strain. The MsERF07 gene improved the plant's water retention capacity and enhanced its drought resistance by increasing the content of plant osmotic regulating substances and the activity of antioxidant enzymes to slow down membrane oxidative damage.

Claims

1. The application of MsERF07 gene in regulating plant drought resistance, characterized in that: The precursor sequence of the MsERF07 gene is shown in SEQ ID No.1, and the mature sequence of the MsERF07 gene is shown in SEQ ID No.

2.

2. The use of the MsERF07 gene in regulating plant drought resistance according to claim 1, characterized in that: When the MsERF07 gene is amplified, MsERF07-F and MsERF07-R are included; wherein the nucleotide sequence of MsERF07-F is shown as SEQ ID No.3; and the nucleotide sequence of MsERF07-R is shown as SEQ ID No.

4.

3. The use of the MsERF07 gene in regulating plant drought resistance according to claim 1, characterized in that: When the MsERF07 gene is overexpressed, the drought resistance of plants is improved.

4. The use of the MsERF07 gene in regulating plant drought resistance according to claim 1, characterized in that: A recombinant vector for overexpressing the MsERF07 gene was constructed using homologous recombination technology, and the recombinant vector was transferred into plants to improve the drought resistance of the plants.

5. The use of the MsERF07 gene in regulating plant drought resistance according to claim 4, characterized in that: The method of using homologous recombination technology to construct a recombinant vector for overexpressing the MsERF07 gene specifically includes digesting the target fragment MsERF07 and the expression vector with KpnⅠ and SalⅠ at 37°C, and then performing a homologous recombination reaction with a homologous recombinase to form a recombinant vector for overexpressing the MsERF07 gene.

6. The use of the MsERF07 gene in regulating plant drought resistance according to claim 5, characterized in that: The overexpression vector is a pCAMBIA1300-35S-EGFP empty vector.

7. The use of the MsERF07 gene in regulating plant drought resistance according to claim 1, characterized in that: The MsERF07 gene improves the water retention capacity of plants and enhances their drought resistance by increasing the content of plant osmotic regulating substances and the activity of antioxidant enzymes to slow down membrane oxidation damage.

8. The use of the MsERF07 gene in regulating plant drought resistance according to claim 1, characterized in that: The application is specifically: using Agrobacterium-mediated method to introduce MsERF07 gene into the plant whose drought resistance is to be improved to obtain transgenic plant.

9. Use of the MsERF07 gene according to any one of claims 1 to 8 in regulating plant drought resistance, characterized in that: The plant of choice is alfalfa.

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