A method for analyzing salt stress of an alfalfa gene promoter

Abstract

The present invention belongs to the technical field of biological genetic engineering, and particularly relates to a method for analyzing the salt stress of an alfalfa gene promoter. The promoter is cloned from the genome of Medicago truncatula variety R108 treated with salt stress through PCR technology. By analyzing the elements of the promoter, it is found that there are multiple cis-acting elements related to adversity stress distributed therein. The present invention constructs a plant expression vector of the reporter gene GUS using the promoter MsPro1, transforms plants through the Agrobacterium infection method, and obtains transgenic Arabidopsis thaliana. Experiments confirm that under salt treatment conditions, the expression activity of the reporter gene GUS in transgenic Arabidopsis thaliana is significantly higher than that of the control, indicating that the promoter MsPro1 is a promoter induced by salt stress. This promoter can be applied to the research of plant salt tolerance genes or salt tolerance gene engineering breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of biological gene engineering, and particularly relates to a method for analyzing salt stress of an alfalfa gene promoter. Background Art

[0002] Alfalfa is a perennial herbaceous legume that contains a variety of minerals and vitamins essential for animal growth, with a protein content of 17% to 24%. Alfalfa (Medicago sativa) boasts a wide cultivation area, a long history, good adaptability, and a rich nutritional profile. It is primarily cultivated in advantageous industrial regions such as Northwest, Northeast, North, and Central my country. Medicago truncatula is the closest known legume relative to alfalfa. Due to its known genome sequence and well-annotated gene functions, it has been widely used as a model species for the analysis of plant gene biological functions and for the genetic breeding of legume forages. In my country, cultivation of alfalfa, a legume forage, is primarily concentrated in northern China. To improve land utilization, saline-alkali lands in the Huanghuaihai region are widely used for alfalfa cultivation. Salt stress typically causes plants to suffer from ionic, osmotic, secondary, and oxidative stresses. Plants grown in saline soils experience high osmotic stress, ion toxicity, and nutrient imbalance, which directly lead to a decline in plant productivity, thereby reducing crop yields and forage quality. Therefore, discovering salt-tolerance genes and their promoters in Medicago truncatula is of great significance. Summary of the invention

[0003] The purpose of the present invention is to provide a method for analyzing salt stress of alfalfa gene promoter.

[0004] The technical solution adopted by the present invention to solve the technical problem is: a method for analyzing salt stress of alfalfa gene promoter, including mining of salt-resistant gene promoter, vector construction and Agrobacterium transformation;

[0005] Vector construction and Agrobacterium transformation include the following steps:

[0006] (1) DNA extraction: DNA was extracted from soil-grown Medicago truncatula seedlings;

[0007] (2) performing PCR amplification using a prepared first solution containing template DNA;

[0008] (3) Gel recovery: Recover the target fragment using a gel recovery kit;

[0009] (4) Homologous recombination ligation, transformation and colony PCR identification:

[0010] a. Perform homologous recombination ligation on the prepared second solution with the gel extraction product. React the second solution at 37 °C for 30 min, then cool it down to 4 °C or immediately place it on ice for cooling;

[0011] b. Transform Escherichia coli competent cells and perform colony PCR identification using the third solution;

[0012] (5) Plasmid extraction and restriction enzyme verification. According to the colony PCR results, perform shaking culture in the fourth solution, extract the plasmid, and perform restriction enzyme verification;

[0013] (6) Select colonies for sample submission and sequencing according to the restriction enzyme digestion results;

[0014] (7) Transform the plasmid into Agrobacterium competent cells. Select the Escherichia coli with correct sequencing results to extract the plasmid for transforming Agrobacterium competent cells;

[0015] (8) Arabidopsis transformation and screening:

[0016] a. Inoculate the verified Agrobacterium into 100 ml of YEP liquid medium and incubate it overnight at 28 °C and 220 rpm in a constant temperature shaker;

[0017] b. When the OD600 value of the bacterial solution is between 0.8 and 1.5, transformation can be carried out;

[0018] c. Centrifuge at 4000 rpm for 15 min at room temperature to collect the bacterial cells, and prepare an equal volume of resuspension solution to resuspend the bacterial cells;

[0019] d. Tilt the Arabidopsis plant so that the inflorescence is immersed in the petri dish containing the bacterial solution, soak for 2 min and then take it out;

[0020] e. Cover the transformed plant with a black plastic bag and remove it the next day;

[0021] f. Screening of homozygous Arabidopsis. After harvesting the seeds of the Arabidopsis infected with Agrobacterium, sow them on the MS plate containing 20 mg / l of Basta, select the resistant plants for transplanting, harvest the seeds of each single plant, and detect the offspring until the homozygous transgenic Arabidopsis is screened out;

[0022] (9) Salt stress treatment and GUS staining:

[0023] Harvest the seeds of the verified lines, dry them in the sun and then sow the seeds on the MS plate containing 20 mg / l of Basta. After growing for 10 days, transplant the seedlings to the MS plates containing different concentrations of NaCl. After 7 days, take the seedlings for GUS staining. The GUS staining color of the Arabidopsis treated with different concentrations of NaCl changes to different degrees with the change of salt concentration. Thus, the cloned promoter MsPro1 is induced and regulated by salt stress.

[0024] Specifically, the mining of the salt-resistant gene promoter includes the following steps:

[0025] a. Treat the cultivated Medicago truncatula R108 with an appropriate concentration of NaCl for 0 hours, 1 hour, 6 hours, and 12 hours. Respectively take the above-ground part and the underground part for transcriptome sequencing.

[0026] b. Analyze the RNA-seq sequencing results of Medicago truncatula R108. Using the treatment of R108 with 0.5% sodium chloride solution for 0 hours as a control, screen out the genes whose expression levels have changed after treating R108 with 0.5% sodium chloride solution for 1 hour, 6 hours, and 12 hours. Then design primers to clone the promoters of the 13 screened genes and construct a salt-resistant gene engineering vector.

[0027] Specifically, step (1) includes the following steps:

[0028] a. Preheat CTAB at 65°C.

[0029] b. Take 1 - 3 g of plant material and grind it in liquid nitrogen until the powder adheres to the side wall of the mortar.

[0030] c. Add the preheated 15 ml of CTAB and 150 μl of mercaptoethanol reducing agent to a 50 ml centrifuge tube and mix well.

[0031] d. Add the ground plant material to the centrifuge tube and mix well. Incubate in a water bath at 65°C for 60 - 90 min, shaking once every 10 min.

[0032] e. Cool to room temperature, add 10 ml of chloroform octanol, extract for 10 min, weigh and balance, centrifuge at 10000 rpm at room temperature for 10 min, take the supernatant, and repeat the extraction once.

[0033] f. Pipette 10 ml of the supernatant, add 400 μl of 5 mol / L NaCl, and mix well.

[0034] g. Add 2.5 volumes of absolute ethanol and mix well to produce a flocculent precipitate.

[0035] h. Use a cut blue pipette tip to aspirate the large flocculent precipitate into a 10 ml centrifuge tube, then add 75% absolute ethanol, and let it stand on ice for 15 - 20 min.

[0036] i. Put the remaining precipitate in the 50 ml centrifuge tube into a -20°C refrigerator for 10 min, then centrifuge for 10 min, pour out the absolute ethanol, wash the precipitate with 75% ethanol, and finally put it into a 10 ml centrifuge tube.

[0037] j. Aspirate the liquid in the centrifuge tube, add 75% ethanol again to wash the precipitate, then add absolute ethanol to rinse, pour out the absolute ethanol and air-dry the precipitate;

[0038] k. Dissolve with 100 μl TE. If the detection quality is poor or the DNA is viscous, add another 900 μl TE to the tube and continue with the following steps:

[0039] l. After adding TE, place it in a 4°C refrigerator. After complete dissolution, add 10 μl RNase, place it in a 37°C water bath for 30 min to digest the RNA;

[0040] m. After taking it out from the water bath, add 1 ml chloroform-octanol, extract for 10 minutes, and centrifuge at 10000 rpm for 10 minutes;

[0041] n. Use a cut blue pipette tip to aspirate about 800 μl of the supernatant and dispense it into two 2 ml centrifuge tubes;

[0042] o. Add 2.5 volumes of absolute ethanol, place it at -20°C to precipitate for 20 minutes, take it out and centrifuge at 10000 rpm for 10 min, pour out the absolute ethanol, and wash the precipitate twice with 1.5 ml of 75% ethanol;

[0043] P. Pour out the 75% ethanol, add absolute ethanol to rinse and then pour out, air-dry the precipitate;

[0044] q. Dissolve with 100 μl TE to extract the DNA.

[0045] Specifically, step (7) includes the following steps:

[0046] a. Place the Agrobacterium competent cells stored at -83 to -78°C at room temperature or hold them between fingertips for a moment. After partial melting, insert them on ice;

[0047] b. Add the target plasmid, gently mix, and let it stand on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min in sequence;

[0048] c. Add 700 μl of sterile liquid medium without antibiotics to the centrifuge tube, mix well, and resuscitate at 28°C and 200 rpm for 2 - 3 h;

[0049] d. Aspirate different volumes of the resuscitation solution and spread them evenly on the YEP plate containing antibiotics. Invert the plate and place it in a 28°C incubator for 2 - 3 days;

[0050] e. Pick monoclonal colonies for bacterial liquid PCR verification.

[0051] The present invention has the following beneficial effects: The nucleotide sequence of the promoter is cloned from the genome of Medicago truncatula variety R108 treated with salt stress by PCR technology; By analyzing the elements of the promoter, it is found that there are multiple cis-acting elements related to stress tolerance distributed therein; A plant expression vector of the reporter gene GUS is constructed using the promoter MsPro1, and plants are transformed by the Agrobacterium infection method to obtain transgenic Arabidopsis thaliana; Experiments confirm that under salt treatment conditions, the expression activity of the reporter gene GUS in transgenic Arabidopsis thaliana is significantly higher than that of the control, indicating that the promoter MsPro1 is a promoter induced by salt stress; This promoter can be applied to the research of plant salt tolerance genes or salt tolerance genetic engineering breeding. Brief Description of the Drawings

[0052] Figure 1 It is a GUS staining analysis diagram of untransformed control Arabidopsis thaliana under different concentrations of salt stress treatment conditions.

[0053] Figure 2 It is a GUS staining analysis diagram of transgenic Arabidopsis thaliana under different concentrations of salt stress treatment conditions. Detailed Description of the Invention

[0054] The present invention will now be described in further detail.

[0055] The coding sequence and application of the promoter MsPro1 of the alfalfa gene MTR_1g077660 that responds to light, abscisic acid, gibberellin, auxin and salt stress. The nucleotide sequence of this promoter is shown in SEQ ID NO.1, and it is cloned from the genome of Medicago truncatula variety R108 treated with salt stress by PCR technology.

[0056] A method for analyzing salt stress of a promoter of an alfalfa gene, comprising the following steps:

[0057] 1. Mining of salt-tolerant genes

[0058] First, cultivate Medicago truncatula R108 to a suitable size, and treat it with an appropriate concentration of sodium chloride for 0 hours, 1 hour, 6 hours, and 12 hours. Then, take the above-ground and underground parts respectively for transcriptome sequencing. Analyze the RNA-seq sequencing results of Medicago truncatula R108. Using the 0.5% sodium chloride solution to treat R108 with 0 hour as the control, screen out the genes with up-regulated expression levels after treating R108 with the 0.5% sodium chloride solution for 1 hour, 6 hours, and 12 hours, which are MTR_4g035905, MTR_0200s0050, MTR_5g064550, MTR_7g103390, and MTR_5g022970 respectively. The genes with down-regulated expression levels are MTR_7g099265, MTR_1g077660, MTR_2g087430, MTR_3g070210, MTR_4g068340, MTR_4g083340, MTR_5g074500, MTR_2g081770, MTR_2g101920, and MTR_8g074570 respectively. Among them, the promoter sequences of MTR_0200s0050 and MTR_7g099265 were not found on NCBI. Therefore, a total of 13 pairs of promoter primers were designed to clone the promoters of the above genes and construct a salt-resistant gene engineering vector.

[0059] 2. Vector construction and Agrobacterium transformation

[0060] (1) DNA extraction: Extract DNA from the soil-cultivated seedlings of Medicago truncatula, including the following steps:

[0061] a. Preheat 2×CTAB at 65°C.

[0062] b. Take the plant material (1 - 3 g) and grind it in liquid nitrogen until the powder on the side wall of the mortar falls in flakes.

[0063] c. Add 15 ml of preheated CTAB and 150 μl of mercaptoethanol reducing agent into a 50 ml centrifuge tube and mix well.

[0064] d. Add the ground material into the centrifuge tube and mix well, then incubate in a water bath at 65°C for 60 - 90 min, shaking once every 10 min (pay attention to venting).

[0065] e. Cool to room temperature, add 10 ml of chloroform octanol, extract for 10 min; weigh and balance, centrifuge at 10000 rpm at room temperature for 10 min, take the supernatant, and repeat the extraction once.

[0066] f. Pipette a certain volume of supernatant (usually 10 ml), add 400 μl of 5 mol / L NaCl, and mix well.

[0067] g. Add 2.5 times the volume of absolute ethanol and mix well to produce a flocculent precipitate.

[0068] h. Use a blue pipette tip with a cut end to aspirate large flocculent precipitates into a 10-ml centrifuge tube (use the pipette tip to pick the precipitate onto the tube wall and aspirate the excess solution), then add 75% absolute ethanol and let it stand on ice for 15 - 20 min.

[0069] i. Put the remaining precipitate in the 50-ml centrifuge tube into a -20°C refrigerator for 10 min, then centrifuge for 10 min, pour out the absolute ethanol, add 75% ethanol to wash the precipitate (twice), and finally put it into a new 10-ml centrifuge tube.

[0070] j. Aspirate the liquid in the centrifuge tube, add 75% ethanol again to wash the precipitate, then add absolute ethanol to rinse, pour out the absolute ethanol and let the precipitate dry.

[0071] k. Dissolve with 100 μl TE (overnight at 4°C, and detect the DNA quality by electrophoresis the next day).

[0072] (The amount of material taken this time is about 1.5 g. If the amount of material is different, the reagent dosage should be changed proportionally).

[0073] If the detected quality is not good (or the DNA is viscous), add another 900 μl of TE to the tube and continue with the following steps:

[0074] l. After adding TE, place it in a 4°C refrigerator. After complete dissolution (no obvious lumps), add 10 μl of RNase, place it in a 37°C water bath for 30 min to digest the RNA.

[0075] m. After taking it out of the water bath, add 1 ml of chloroform-octanol, extract for 10 minutes, and centrifuge at 10,000 rpm for 10 minutes.

[0076] n. Use a blue pipette tip with a cut end to aspirate about 800 μl of the supernatant and dispense it into two 2-ml centrifuge tubes.

[0077] o. Add 2.5 times the volume of absolute ethanol, place it at -20°C to precipitate for 20 minutes, take it out and centrifuge at 10,000 rpm for 10 minutes, pour out the absolute ethanol, and wash the precipitate twice with 1.5 ml of 75% ethanol.

[0078] P. Pour out the 75% ethanol, add absolute ethanol to rinse and then pour out, and let the precipitate dry.

[0079] q. Dissolve with 100 μl TE.

[0080] (2) PCR amplification

[0081] System:

[0082]

[0083]

[0084] Procedure:

[0085]

[0086] (3) Gel recovery: Recover the target fragment using a gel recovery kit.

[0087] (4) Homologous recombination ligation, transformation and colony PCR

[0088] Homologous recombination ligation (Novoprotein) system:

[0089]

[0090] React at 37 °C for 30 min; cool to 4 °C or immediately place on ice. Transform Escherichia coli competent cells and perform colony PCR identification.

[0091] Colony PCR:

[0092] System:

[0093]

[0094] Procedure:

[0095]

[0096]

[0097] (5) Plasmid extraction and restriction enzyme verification: According to the colony PCR results, perform shaking culture, extract the plasmid, and perform restriction enzyme verification.

[0098]

[0099] Incubate in a 37 °C metal bath for 30 min.

[0100] (6) Select colonies according to the restriction enzyme digestion results and send them for sequencing.

[0101] (7) Transformation of plasmid into Agrobacterium competent cells: Select Escherichia coli with correct sequencing results to extract the plasmid for transformation of Agrobacterium competent cells:

[0102] a. Place the Agrobacterium competent cells stored at -83 to -78 °C at room temperature or hold them between your fingertips for a moment. After partial thawing, insert them on ice.

[0103] b. Add the target plasmid, gently mix, and incubate on ice for 5 min, in liquid nitrogen for 5 min, in a 37 °C water bath for 5 min, and in an ice bath for 5 min in sequence.

[0104] c. Add 700 μl of sterile liquid medium (YEP) without antibiotics to the centrifuge tube. After mixing, resuscitate at 28 °C and 200 rpm for 2 - 3 h.

[0105] d. According to the experimental needs, pipette different volumes of the resuscitation solution and evenly spread it on the YEP plate containing antibiotics (kan + rif). Invert the plate and place it in an incubator at 28 °C for 2 - 3 days.

[0106] e. Pick monoclonal colonies for verification by bacterial liquid PCR.

[0107] 3. Arabidopsis transformation and screening

[0108] a. Inoculate the verified Agrobacterium (GV3101) into 100 ml of YEP liquid medium (kan + rif) and incubate it overnight at 28 °C and 250 rpm on a constant temperature shaker.

[0109] b. When the OD600 value of the bacterial liquid is between 0.8 - 1.5, transformation can be carried out. The transformation effect is the best when OD600 is around 1.2.

[0110] c. Centrifuge at 4000 rpm for 15 min at room temperature to collect the bacterial cells.

[0111] d. Prepare an equal - volume resuspension solution to resuspend the bacterial cells (100 ml)

[0112] Sucrose 5% 5 g

[0113] Silwet L - 77 0.02% 20 μl

[0114] e. Tilt the Arabidopsis plant so that the inflorescence is immersed in the petri dish containing the bacterial liquid. After soaking for 2 min, take it out.

[0115] f. Cover the transformed plants with a black plastic bag and remove it the next day.

[0116] g. Screening of homozygous Arabidopsis: After harvesting the seeds of Arabidopsis infected with Agrobacterium, sow them on the MS plate containing Basta (20 mg / l). Select resistant plants for transplanting, harvest seeds from individual plants, and detect the offspring until homozygous transgenic Arabidopsis is screened out.

[0117] 3. Salt stress treatment and GUS staining

[0118] a. Harvest the seeds of the verified lines, dry them in the sun, and then sow the seeds on the MS plate (containing 20 mg / l Basta). After growing for 10 days, transplant the seedlings to the MS plates containing different concentrations of NaCl (0 mM, 50 mM, 100 mM, 150 mM). After 7 days, take the seedlings for GUS staining, and the results are as Figure 2As shown, compared with MS, the GUS staining color of transgenic Arabidopsis thaliana treated with 50 mM NaCl, 100 mM NaCl, and 150 mM NaCl salts changed to varying degrees with the change of salt concentration. For example, Figure 1 , no GUS staining appeared in the non-transgenic control Arabidopsis thaliana under different degrees of salt stress treatment, indicating that the cloned promoter MsPro1 was indeed induced and regulated by salt stress.

[0119] The present invention is not limited to the above embodiments. Anyone should know that structural changes made under the inspiration of the present invention, as long as they have the same or similar technical solutions as the present invention, all fall within the protection scope of the present invention.

[0120] The technologies, shapes, and structures not described in detail in the present invention are all well-known technologies.

[0121] SEQ ID NO.1

[0122]

Claims

1. A salt stress-induced alfalfa gene promoter MsPro1, characterized in that, The nucleotide sequence of the promoter is shown as SEQ ID NO.

1.

2. The salt stress-induced alfalfa gene promoter MsPro1 according to claim 1, characterized in that, It is obtained through the steps of mining the salt-tolerant gene promoter, vector construction, and Agrobacterium transformation. Vector construction and Agrobacterium transformation include the following steps: (1) DNA extraction: Extract DNA from the soil-grown seedlings of Medicago truncatula. (2) Perform PCR amplification with the first solution containing template DNA prepared. (3) Gel recovery: Recover the target fragment using a gel recovery kit. (4) Homologous recombination ligation, transformation, and colony PCR identification: a. Perform homologous recombination ligation on the second solution containing the gel recovery product. The second solution reacts at 37 °C for 30 min, then cools to 4 °C or is immediately placed on ice. b. Transform Escherichia coli competent cells and perform colony PCR identification with the third solution. (5) Plasmid extraction and enzyme digestion verification: According to the colony PCR results, perform shaking culture in the fourth solution, extract the plasmid, and perform enzyme digestion verification. (6) Select colonies for sequencing according to the enzyme digestion results. (7) Transform the plasmid into Agrobacterium competent cells. Select the Escherichia coli with correct sequencing results to extract the plasmid for transforming Agrobacterium competent cells. (8) Arabidopsis transformation and screening: a. Inoculate the verified Agrobacterium into 100 ml of YEP liquid medium and incubate overnight at 28 °C and 220 rpm in a constant temperature shaker. b. When the OD600 value of the bacterial solution is between 0.8 and 1.5, transformation can be carried out. c. Centrifuge at 4000 rpm for 15 min at room temperature to collect the bacteria, and configure an equal volume of resuspension solution to resuspend the bacteria. d. Tilt the Arabidopsis plant so that the inflorescence is immersed in the petri dish containing the bacterial solution, soak for 2 min and then take out. e. Cover the transformed plant with a black plastic bag and remove it the next day. f. Screening of homozygous Arabidopsis: After harvesting the seeds of Arabidopsis infected with Agrobacterium, sow them on MS plates containing 20 mg / l Basta, select resistant plants for transplanting, harvest seeds from single plants, and detect the offspring until the transgenic Arabidopsis homozygote is screened out. (9) Salt stress treatment and GUS staining: Harvest the seeds of the verified lines, dry them in the sun and then sow them on MS plates containing 20 mg / l Basta. After growing for 10 days, transplant the seedlings to MS plates containing different concentrations of NaCl. After 7 days, take the seedlings for GUS staining. The GUS staining color of Arabidopsis treated with different concentrations of NaCl changes to varying degrees with the change of salt concentration. The promoter MsPro1 cloned thus is induced and regulated by salt stress.

3. The salt stress-induced alfalfa gene promoter MsPro1 according to claim 2, characterized in that, The mining of the salt-tolerant gene promoter includes the following steps: a. Treat the cultured Medicago truncatula R108 with appropriate concentrations of NaCl for 0 hours, 1 hour, 6 hours, and 12 hours, respectively. Take the above-ground and underground parts and perform transcriptome sequencing. b. Analyze the RNA-seq sequencing results of Medicago truncatula R108. Use the treatment of R108 with 0.5% sodium chloride solution for 0 hours as a control, screen out the genes whose expression levels change after R108 is treated with 0.5% sodium chloride solution for 1 hour, 6 hours, and 12 hours, and then design primers to clone the promoters of the 13 selected genes to construct a salt-resistant gene engineering vector.

4. A salt stress-induced alfalfa gene promoter MsPro1 according to claim 2, characterized in that Step (1) includes the following steps: a. Preheat CTAB at 65 °C. b. Take 1 - 3 g of plant material and grind it in liquid nitrogen until the powder adheres to the side wall of the mortar. c. Add the preheated 15 ml of CTAB and 150 μl of mercaptoethanol reducing agent to a 50 ml centrifuge tube and mix well. d. Add the ground plant material to the centrifuge tube and mix well, incubate in a water bath at 65 °C for 60 - 90 min, and shake once every 10 min. e. Cool to room temperature, add 10 ml of chloroform - octanol, extract for 10 min, weigh and balance, centrifuge at 10000 rpm at room temperature for 10 min, take the supernatant, and repeat the extraction once. f. Pipette 10 ml of the supernatant, add 400 μl of 5 mol / L NaCl, and mix well. g. Add 2.5 volumes of absolute ethanol and mix well to produce a flocculent precipitate. h. Use a cut blue pipette tip to aspirate the large flocculent precipitate into a 10 ml centrifuge tube, then add 75% absolute ethanol, and let it stand on ice for 15 - 20 min. i. Put the remaining precipitate in the 50 ml centrifuge tube into a - 20 °C refrigerator for 10 min, then centrifuge for 10 min, pour out the absolute ethanol, add 75% ethanol to wash the precipitate, and finally put it into a 10 ml centrifuge tube. j. Aspirate the liquid in the centrifuge tube, add 75% ethanol again to wash the precipitate, then add absolute ethanol to rinse, pour out the absolute ethanol and air - dry the precipitate. k. Dissolve with 100 μl of TE. If the detected quality is not good or the DNA is viscous, add another 900 μl of TE to the tube and continue with the following steps: l. After adding TE, place it in a 4 °C refrigerator. After complete dissolution, add 10 μl of RNase, place it in a 37 °C water bath for 30 min to digest the RNA. m. After taking it out of the water bath, add 1 ml of chloroform - octanol, extract for 10 minutes, and centrifuge at 10000 rpm for 10 minutes. n. Use a cut blue pipette tip to aspirate about 800 μl of the supernatant and dispense it into two 2 ml centrifuge tubes. o. Add 2.5 volumes of absolute ethanol, place it at - 20 °C to precipitate for 20 minutes, take it out and centrifuge at 10000 rpm for 10 min, pour out the absolute ethanol, and wash the precipitate twice with 1.5 ml of 75% ethanol. P. Pour out the 75% ethanol, add absolute ethanol to rinse and then pour out, air - dry the precipitate. q. Dissolve with 100 μl of TE to extract the DNA.

5. The salt stress-induced alfalfa gene promoter MsPro1 according to claim 2, characterized in that, Step (7) includes the following steps: a. Place the Agrobacterium tumefaciens competent cells stored at - 83 - - 78 °C at room temperature or hold them between fingertips for a moment. After partial melting, insert them on ice. b. Add the target plasmid, mix gently, and let it stand on ice for 5 min, in liquid nitrogen for 5 min, in a 37 °C water bath for 5 min, and in an ice bath for 5 min in sequence. c. Add 700 μl of sterile liquid culture medium without antibiotics to the centrifuge tube, mix thoroughly, and incubate at 28°C, 200 rpm for 2-3 hours. d. Pipette different volumes of resuscitation solution and evenly spread it onto a YEP plate containing antibiotics. Place the plate upside down in a 28°C incubator and incubate for 2-3 days. e. Select a single clone and perform bacterial liquid PCR verification.

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