Medicago sativa MsAST gene and application thereof 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 MsAST gene and application thereof in regulation and control of plant salt tolerance. The nucleotide sequence of the medicago sativa MsAST gene is as shown in SEQ ID NO. 1. According to the invention, the salt tolerance-related MsASTT gene is cloned from alfalfa, a recombinant vector of the MsASTT gene is constructed, the function of improving the salt tolerance of plants by the MsASTT gene is verified in arabidopsis thaliana, and the MsASTT gene can be used for improving the salt tolerance of alfalfa and creating an alfalfa germplasm material with strong salt tolerance.

Description

Technical Field

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

[0002] Alfalfa (Medicago sativa.L) is a perennial leguminous herb and one of the most important forage crops in the world. Alfalfa has a long cultivation history in China and is mainly distributed in the northeast, north, northwest and other regions, with strong drought resistance, cold resistance and salt-alkali tolerance. Its main root is developed and has strong nitrogen fixation ability, which can loosen the soil, promote the accumulation of organic matter and total nitrogen, improve the soil structure and enhance the soil fertility. Alfalfa has high yield, good palatability, high protein content, is rich in various amino acids necessary for animal growth and development, contains trace elements such as polysaccharides, flavonoids, calcium and phosphorus, and is a high-quality forage.

[0003] Although alfalfa can withstand a certain degree of salt stress, with the aggravation of soil salinization and the increasing demand for forage in the development of animal husbandry, how to maintain the quality and yield of alfalfa under salt stress and ensure its efficient ecological benefits has become a key problem to be solved urgently. Therefore, exploring the regulatory mechanism of melatonin in alleviating alfalfa salt stress and using molecular biology means to cultivate new alfalfa varieties with high quality, high yield and stress resistance is of great significance for the development of animal husbandry.

[0004] Melatonin is a bioactive molecule that has a significant impact on the circadian rhythm, anti-aging and other aspects of animals. Some studies have shown that melatonin plays a variety of regulatory functions in the process of plant growth and development, and plays an important role in protecting plants from environmental stresses, including high temperature and low temperature, water stress, osmotic and ionic stress, ultraviolet radiation, heavy metal stress, etc. The synthesis of melatonin in plants requires 4 consecutive enzymatic reactions, and N-acetylserotonin methyltransferase (ASMT) is the last key enzyme in the synthesis process. However, there are few reports on the research of the ASMT gene expression regulation mechanism, especially the molecular mechanism of ASMT in response to salt stress in alfalfa is still unclear. Summary of the Invention

[0005] Aiming at the above deficiencies in the prior art, the purpose of the present invention is to provide the Medicago sativa MsASMT gene and its application in regulating plant salt tolerance.

[0006] In order to achieve the above invention purpose, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect, an alfalfa MsASMT gene is provided, and its nucleotide sequence is shown as SEQ ID NO.1.

[0008] In the second aspect, a transgenic vector containing the alfalfa MsASMT gene is provided.

[0009] In the third aspect, a recombinant strain or recombinant cell containing the alfalfa MsASMT gene is provided.

[0010] In the fourth aspect, the application of overexpression of the alfalfa MsASMT gene in improving plant salt tolerance is provided.

[0011] In the fifth aspect, the application of the alfalfa MsASMT gene in improving the resistance quality of alfalfa and breeding excellent salt-tolerant alfalfa varieties is provided.

[0012] The beneficial effects of the present invention are as follows:

[0013] In the present invention, the MsASMTT gene related to salt tolerance is cloned from alfalfa, a recombinant vector of the MsASMT gene is constructed, and the function of the MsASMT gene in improving plant salt tolerance is verified in Arabidopsis thaliana. 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

[0014] Figure 1 It is the gel electrophoresis diagram of the cloning of MsASMT of 'Zhongmu No.1' in the examples;

[0015] Figure 2 It is the electrophoresis gel diagram of the amplification of the homologous recombinant MsASMT gene in the examples;

[0016] Figure 3 It is the gel electrophoresis diagram of the double digestion of the PCF203 plasmid in the examples;

[0017] Figure 4 It is the vector map of the recombinant expression vector PCF203-ASMT in the examples;

[0018] Figure 5 It is the PCR identification gel diagram of the PCF203-MsASMT homologous recombinant plasmid in the examples; Figure 6 It is the PCR identification gel diagram of the Arabidopsis thaliana overexpressing MsASMT; Figure 7 It is the phenotypes of wild-type and Arabidopsis thaliana overexpressing MsASMT under 150 mM NaCl treatment. Detailed Embodiments

[0019] The specific embodiments of the present invention will be described below to facilitate those skilled in the art of the present technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art of the present 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 and creations using the concept of the present invention are within the scope of protection.

[0020] Example

[0021] 1. Experimental procedure

[0022] 1.1 Materials

[0023] 1.1.1 Test materials

[0024] Alfalfa 'Zhongmu No. 1' was planted in the artificial climate chamber of the School of Ecology and Environment, Ningxia University. Fresh leaves were quickly placed in liquid nitrogen and stored in a -80°C refrigerator.

[0025] Arabidopsis thaliana is the Columbia wild type Arabidopsis thaliana (Columbia-0, WT).

[0026] The overexpression vector is the vector PCF203 stored in the laboratory.

[0027] 1.1.2 Instruments and reagents

[0028] Reagents: DNA Marker, Loading Buffer, Taq Master mix were purchased from Takara; BamH I, Sac I were purchased from NEB; reverse transcription kit, high-fidelity enzyme were purchased from Novizan; RNA extraction kit, DNA purification and recovery kit, and plasmid extraction kit were purchased from TIANGEN; competent cells DH5α, GV3101 were purchased from Weidi Biology; blunt-end vector was purchased from Aidlab; homologous recombination enzyme Seamless Cloning and Assembly Kit was purchased from Beijing TransGen Biotech Co., Ltd.

[0029] Instruments: laminar flow hood, electrophoresis tank, electrophoresis apparatus, micropipette, electronic balance, high-speed refrigerated centrifuge, PCR instrument, gel imaging system, refrigerator, centrifuge, constant temperature water bath, oven, constant temperature incubator, etc.

[0030] 1.1.3 Culture media

[0031] Table 1-1 Formulas of culture media required for the experiment

[0032]

[0033] 1.1.4 Antibiotics

[0034] KanR(50mg·mL -1 ): Dissolve 0.5 g of kanamycin powder, make up the volume to 10 mL, dispense into 1.5 mL sterile centrifuge tubes, and store at -20 °C.

[0035] Rif(50mg·mL -1 ): Dissolve 0.5 g of rifampicin powder in methanol solution, make up the volume to 10 mL, dispense into 1.5 mL sterile centrifuge tubes, and store at -20 °C.

[0036] Spe(50mg·mL -1 ): Dissolve 0.5 g of spectinomycin powder, make up the volume to 10 mL, dispense into 1.5 mL sterile centrifuge tubes, and store at -20 °C.

[0037] 1.2 Cloning of Medicago sativa MsASMT gene

[0038] 1.2.1 Total RNA extraction and cDNA acquisition of Medicago sativa

[0039] 1.2.1.1 RNA extraction

[0040] 1) Grind about 0.1 g of Medicago sativa leaves with liquid nitrogen. After thorough grinding, add 500 μL of lysis buffer, shake well, and centrifuge at 12,000 rpm for 2 min;

[0041] 2) Use a pipette to transfer the supernatant to a filter column and centrifuge at 12,000 rpm for 2 min; Transfer the liquid to a new centrifuge tube, add anhydrous ethanol to 1.4 times the volume, transfer to an adsorption column and then centrifuge at 12,000 rpm for 30 s, discard the filtrate;

[0042] 3) Add 350 μL of protein removal solution and centrifuge at 12,000 rpm for 30 s, discard the filtrate;

[0043] 4) Add 500 μL of wash buffer and centrifuge at 12,000 rpm, discard the filtrate; Repeat this operation once. Let the centrifuge tube stand at room temperature for 10 min to ensure complete ethanol evaporation. During this period, open the centrifuge tube cap;

[0044] 5) Place the adsorption column in a new enzyme-free centrifuge tube, add 40 μL of enzyme-free ddH2O dropwise (to increase the elution efficiency, ddH2O can be preheated to 50 °C), let it stand for 5 min and then centrifuge at 12,000 rpm for 2 min; Collect the RNA solution and centrifuge again;

[0045] 6) Use a ultra-micro ultraviolet spectrophotometer and gel electrophoresis to check the quality and concentration of RNA.

[0046] 1.2.1.2 cDNA acquisition

[0047] The cDNA was obtained using the Novoprotein Reverse Transcription Kit as follows:

[0048] 1) Denaturation of RNA template

[0049] Table 1-1 Reaction system for RNA template denaturation

[0050]

[0051] Reaction program: React at 65 °C for 5 min and then place on ice after completion.

[0052] 2) Removal of genomic DNA

[0053] Table 1-2 Reaction system for removing genomic DNA

[0054]

[0055] Reaction program: React at 42 °C for 2 min

[0056] 3) Preparation of cDNA synthesis reaction solution

[0057] Table 1-3 cDNA synthesis reaction system

[0058]

[0059]

[0060] Reaction program: React at 37 °C for 45 min and 85 °C for 5 s

[0061] 4) The reaction product cDNA was stored in a -80 °C refrigerator.

[0062] 1.2.2 Amplification of Medicago sativa MsASMT

[0063] 1.2.2.1 Primer design and synthesis

[0064] Based on the ASMT sequence of the Medicago sativa genome, the ASMT sequences of related species were obtained by blast, and primers were designed at the 5' end of the CDS region. The primers were synthesized by BGI. The primers were named ASMT-F1 and ASMT-R1 respectively.

[0065] 1.2.2.2 PCR reaction conditions and system for Medicago sativa MsASMT gene

[0066] The PCR reaction system was 20 μL, and the cDNA obtained in 1.2.1 was used as a template for cloning, and carried out according to the following conditions:

[0067] Table 1-4 MsASMT amplification PCR reaction system

[0068]

[0069] Table 1-5 MsASMT Amplification PCR Reaction Program

[0070]

[0071] 1.2.2.3 Recovery of Target Fragment

[0072] After the PCR reaction, electrophoresis is carried out using 1% agarose gel. After the electrophoresis is completed, the bands are observed in the gel imaging system. The gel with the correct bands is cut off and the DNA purification and recovery kit is used for gel recovery. After measuring the concentration and quality of the gel recovery product, it is stored in a -20°C refrigerator.

[0073] 1.2.2.4 Ligation of Target Fragment

[0074] The target fragment in 1.2.2.3 is ligated with the blunt-ended vector at room temperature. The ligation reaction system is shown in the table:

[0075] Table 1-7 MsASMT Ligation of Blunt-Ended Vector Reaction System

[0076]

[0077] Reaction Program: 28°C for 5 min. After the ligation is completed, transform Escherichia coli.

[0078] 1.2.2.5 Transformation and Identification

[0079] Escherichia coli and the recombinant plasmid are transformed using the heat shock method. Take 5 μL of the recombinant plasmid and add it to the competent Escherichia coli in the frozen-thawed state. Gently flick to mix, react at 0°C for 30 min, heat shock at 37°C for 1.5 min, then place on ice for 3 min. Add 500 μL of LB medium and culture at 180 rpm, 37°C for 1 hour. After the culture is completed, take 20 μL of the bacterial solution and spread it on the LB solid medium containing Spe, and culture it inverted at 37°C for 16 h. The next day, select single colonies for PCR identification and expansion culture.

[0080] Table 1-8 PCR Identification Reaction System

[0081]

[0082] Table 1-9 PCR Identification Reaction Program

[0083]

[0084] For the bacterial solution corresponding to the bright band detected by gel electrophoresis of the PCR reaction solution, it is sent to Sangon Biotech for Sanger sequencing.

[0085] 1.3 Construction of Overexpression Vector by Homologous Recombination

[0086] 1.3.1 Primer Design

[0087] According to the primer design principle, the homologous recombination primers consist of three parts. The upstream primer is composed of the vector restriction site, about 20 bp of the vector sequence before the restriction site, and about 20 bp of the sequence at the 5'-end of the upstream of the target fragment. The downstream primer is composed of the downstream restriction site of the vector, about 20 bp of the vector sequence after the restriction site, and about 20 bp of the sequence at the 5'-end of the downstream of the target fragment. The primers were synthesized by BGI.

[0088] 1.3.2 Amplification of target fragment

[0089] Using the recombinant plasmid obtained in 1.2.1 as a template, the target fragment was amplified. The reaction system and procedure are as follows:

[0090] Table 1-10 PCR reaction system for amplifying MsASMT with homologous arm primers

[0091]

[0092] Table 1-11 PCR reaction procedure for amplifying MsASMT with homologous arm primers

[0093]

[0094]

[0095] The target fragment was purified and recovered according to the method in 1.2.2.3.

[0096] (3) Preparation of linearized vector

[0097] Table 1-12 Double digestion reaction system of PCF203 vector

[0098]

[0099] Digestion procedure: 37°C for 2 h

[0100] The target fragment was purified and recovered according to the method in 1.2.2.3.

[0101] (4) Homologous recombination ligation

[0102] The linearized vector and the target fragment were subjected to a recombination reaction under the action of a recombinase.

[0103] Ligation reaction system:

[0104] Table 1-13 Homologous recombination ligation reaction system

[0105]

[0106] Reaction procedure: 50°C for 20 min

[0107] Transfer the ligation product into competent Escherichia coli cells and culture them overnight for 12 - 16 h in LB medium supplemented with 50 mg / ml Spe. The next day, pick monoclonal colonies for colony PCR identification. Expand the culture of positive clones at 37 °C, extract plasmids, and send them for sequencing.

[0108] 1.4 Transformation of Agrobacterium tumefaciens with the recombinant plasmid

[0109] The method for transforming Agrobacterium tumefaciens with the recombinant plasmid PCF203 - ASMT refers to the method in 1.2.2.5, and the temperature is set at 28 °C. The reaction system and procedure for colony PCR identification are as follows:

[0110] Table 1 - 6 Reaction system for recombinant plasmid identification

[0111]

[0112] Table 1 - 7 Reaction procedure for recombinant plasmid identification

[0113]

[0114] Select 3 positive bacterial solutions with bright bands, mix them, expand the culture, and preserve the bacteria with glycerol at -80 °C.

[0115] 1.5 Infection of Arabidopsis thaliana with Agrobacterium tumefaciens

[0116] 1.5.1 Bacterial solution preparation

[0117] Take 2 μl of the preserved Agrobacterium tumefaciens bacterial solution containing the PCF203 - MsASMT1 plasmid and spread it on an LB plate containing Spec. Culture it at 28 °C for 48 h. After single colonies grow, pick a single colony into 6 ml of LB liquid medium containing Spec and culture it overnight at 200 rpm and 28 °C for 16 h. The next day, transfer it into 40 ml of LB liquid medium containing Spec and shake it vigorously for 6 h until the OD600 of the bacterial solution is between 0.6 and 0.8. Centrifuge at 4000 rpm for 15 min to collect the bacteria. Add the infection solution to resuspend the bacteria for Arabidopsis thaliana infection.

[0118] 1.5.2 Infection

[0119] After wild - type Arabidopsis thaliana grows for about 28 days, it is ready for infection (bolting and having multiple lateral branches). During infection, invert the inflorescence part of Arabidopsis thaliana into the infection solution and soak it for about 1 min. After infecting Arabidopsis thaliana, place it in the dark for 1 day and then move it out to grow normally under light. After 7 days, perform the second infection using the same method. After infection, Arabidopsis thaliana grows for about 2 months to harvest T1 - generation transgenic Arabidopsis thaliana seeds.

[0120] 1.5.3 Screening of T1 - generation Arabidopsis thaliana seeds

[0121] 1.5.3.1 Arabidopsis thaliana Seed Disinfection

[0122] Take an appropriate amount of T1 generation Arabidopsis thaliana seeds and place them in a 2 ml centrifuge tube. Wash with 75% ethanol for 30 s, disinfect with 0.1% mercuric chloride solution for 8 min. After washing and disinfection, add sterile water and repeat washing 4 - 5 times. Let it stand still to precipitate the Arabidopsis thaliana seeds, and discard the liquid. Place the seeds in a 4°C refrigerator for vernalization for 24 h.

[0123] 1.5.3.2 Arabidopsis thaliana Screening

[0124] Inoculate the vernalized T1 generation seeds into a 1 / 2 MS solid medium containing KanR for screening, and place them in a tissue culture room to grow (temperature set at 22°C). Screen out the homozygous Arabidopsis thaliana with KanR resistance, and harvest the seeds to obtain T2 generation transgenic Arabidopsis thaliana.

[0125] 1.5.4 Salt Tolerance Function Verification of Transgenic Arabidopsis thaliana

[0126] After disinfecting the harvested T2 generation Arabidopsis thaliana seeds according to the method of 4.5.3.1, inoculate them on a 1 / 2 MS medium containing KanR. When the Arabidopsis thaliana bud length grows to 1 cm, transfer it to a vertical plate containing 150 mM NaCl, and use clear water as a control to evaluate the salt tolerance of transgenic Arabidopsis thaliana.

[0127] 2 Results and Analysis

[0128] 2.1 MsASMT Gene Cloning and Function Analysis

[0129] 2.1.1 Obtaining the Target Gene

[0130] (1) RNA Quality Identification

[0131] Use the TIANGEN plant RNA extraction kit to extract the RNA of Medicago sativa 'Zhongmu No. 1', and use the Novizan reverse transcription kit to synthesize cDNA. Immediately electrophorese the extracted RNA to obtain clear and bright 28s and 18s bands. To further determine the RNA quality, use an ultra - micro ultraviolet spectrophotometer for quantitative analysis. The results show that the OD260 / 280 value of the sample is about 1.88, and the concentration is 425 ng / μL, indicating that the RNA has no protein contamination and can be used as a reverse transcription template.

[0132] (2) Primer Design

[0133] Table 2 - 1 Primers for Medicago sativa MsASMT Cloning

[0134]

[0135] (3) Gene Cloning

[0136] The amplification template of MsASMT was the cDNA of Medicago sativa 'Zhongmu No. 1' obtained by reverse transcription. The product was detected by 1% agarose gel electrophoresis, and the band size was about 1500 bp. The results were as Figure 1 , which were in line with the expected results. The target gene was recovered from the gel, ligated to a blunt-ended vector, transformed into Escherichia coli, and the bacterial liquid was sent to Sangon Biotech for sequencing. The CDS sequence of the MsASMT gene of Medicago sativa was obtained and registered on NCBI (accession number: PP915624.1).

[0137] 2.2 Construction of homologous recombination vector

[0138] 2.2.1 Design of recombinant primers and gene cloning

[0139] Table 2-2 Primers for constructing recombinant expression vector

[0140]

[0141] Using the cDNA of Medicago sativa 'Zhongmu No. 1' as the template and the sequences shown in the table as primers, the target fragment with homologous sequences was amplified. In the figure, DL2000 bp marker was used, and the target genes were amplified in lanes m1-4. The bands were clear and bright, indicating that the obtained target genes ( Figure 2 ).

[0142] 2.2.2 Preparation of linearized vector

[0143] In this experiment, the plant expression vector PCF203 plasmid stored in the laboratory was used, and double digestion was performed with the restriction enzymes BamH I and Sac I from New England Biolabs. The digestion results were as Figure 3 shown. The linearized vector was recovered by combining and gel-extracting the bands in lanes 1, 2, and 3.

[0144] 2.2.3 Ligation of vector and target fragment and identification of recombinant plasmid

[0145] Under the action of highly efficient homologous recombination enzyme, the digested vector and the target fragment with 20-25 bp overlapping regions at both ends of the gene were directionally recombined to achieve seamless splicing. The picture of the recombinant plasmid vector was as Figure 4 shown. The recombinant plasmid was transformed into Escherichia coli according to the method in 1.2.5, and positive colonies were picked for shaking culture. At the same time, colony PCR ( Figure 5 ) was performed. The bacterial liquids corresponding to the positive colonies in lanes 1, 2, 4, 5, and 8 were expanded and cultured, and then the plasmids were extracted and sent to Sangon Biotech for sequencing. After successful sequencing, the correct plasmid was transformed into Agrobacterium for subsequent experiments.

[0146] 2.3 Functional analysis of salt tolerance of transgenic MsASMT Arabidopsis

[0147] The overexpression vector pCF203-MsASMT was transferred into Arabidopsis thaliana by the Agrobacterium-mediated method. T0 generation seeds were collected and screened on 1 / 2 MS medium containing kanamycin to obtain T1 generation transgenic Arabidopsis thaliana. Plant genomic DNA was extracted for PCR amplification detection. The screening and identification results of positive seedlings showed that a total of 5 overexpression lines were obtained ( Figure 6 ). The offspring of these 5 lines were screened, and finally, homozygous plants of 2 transgenic lines were obtained. The homozygous transgenic Arabidopsis thaliana obtained was subjected to a salt tolerance functional phenotype analysis.

[0148] Arabidopsis thaliana seeds were sown on 1 / 2 MS medium. When the root length of the seedlings grew to about 1 cm, wild-type and transgenic seedlings with consistent root lengths were selected and transferred to a vertical plate of 1 / 2 MS medium. A 150 mM NaCl solution was added to the medium, and the growth trend was observed. The primary root length was measured at 7 days of growth ( Figure 7 ). The results showed that: on the medium without stress, the growth of WT and MsASMT transgenic lines was consistent, with no significant difference; under the treatment of 150 mM NaCl, the growth of wild-type Arabidopsis thaliana was inhibited, and the primary root length was significantly shorter than that of the transgenic lines. The primary root length decreased by 49.44% compared with that without treatment, the leaves turned yellow, curled, and wilted significantly, while the leaves of the transgenic lines showed light green and grew well, and the primary root length decreased by 41.72% compared with that without treatment (Table 2-3). These results indicate that MsASMT1 transgenic Arabidopsis thaliana seedlings have stronger salt tolerance than wild-type Arabidopsis thaliana.

[0149] Table 2-3 Determination of Arabidopsis thaliana root length

[0150]

[0151] The synthesis of melatonin in plants requires 4 consecutive enzymatic reactions. N-acetylserotonin methyltransferase (ASMT) is the last key enzyme in the synthesis process. This gene is significantly induced and expressed under stress conditions such as leaf senescence, heavy metals, and herbicides. High temperature and darkness can also increase the enzyme activity of ASMT and thus increase the synthesis of melatonin, which plays an important role in enhancing the stress resistance of plants. In this invention, the MsASMTT gene related to salt tolerance was cloned from Medicago sativa, a recombinant vector of the MsASMT gene was constructed, and the function of the MsASMT gene in improving plant salt tolerance was verified in Arabidopsis thaliana. This invention can be used to improve the salt tolerance of alfalfa and create germplasm materials of Medicago sativa with strong salt tolerance.

[0152] 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 any aspect, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

[0153] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment contains only one independent technical solution. This narrative style of the specification is only for clarity. Those skilled in the art should take the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. The Medicago sativa MsASMT gene, characterized in that, Its nucleotide sequence is shown as SEQ ID NO.

1.

2. A transgenic vector containing the alfalfa MsASMT gene described in claim 1.

3. A recombinant strain or recombinant cell containing the alfalfa MsASMT gene described in claim 1.

4. Use of overexpression of the alfalfa MsASMT gene described in claim 1 in improving plant salt tolerance.

5. Use of the alfalfa MsASMT gene described in claim 1 in improving the resistance quality of alfalfa and breeding excellent salt-tolerant alfalfa varieties.

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