CN116987163A

CN116987163A - Stress-resistance related protein MsWRKY2G, and coding gene and application thereof

Info

Publication number: CN116987163A
Application number: CN202310967061.4A
Authority: CN
Inventors: 刘志鹏; 董书伟; 毛培; 罗栋; 周强
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2023-08-02
Filing date: 2023-08-02
Publication date: 2023-11-03

Abstract

The invention discloses a stress-resistance related protein MsWRKY2G, and a coding gene and application thereof. The expression quantity of the protein is increased under drought conditions, and the encoded protein is positioned to the cell nucleus; secondly, the MsWRKY2G gene can improve the drought resistance of the alfalfa on the premise of not changing the yield and quality of the alfalfa, and is beneficial to cultivating new varieties of the alfalfa with strong drought resistance and expanding the planting scale of the alfalfa. The invention not only provides a research thought for researching drought-resistant genes on a molecular level, but also provides a theoretical basis for improving biomass and quality of pasture crops under drought stress.

Description

Stress-resistance related protein MsWRKY2G, and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to stress-resistance related protein MsWRKY2G, and a coding gene and application thereof.

Background

Alfalfa (Medicago sativa l.) is known as "forage king" and is one of the most important forage crops worldwide due to its high yield, high quality, good palatability and broad adaptability. The alfalfa planting areas in China are mainly concentrated in northwest, northeast, north China arid and semiarid areas, and alfalfa belongs to high-water-consumption pasture, and a large amount of water is needed in the growing and developing process. Thus, drought has become one of the major factors limiting alfalfa yield and quality and plant area.

Drought stress is a multidimensional stress that causes tremendous changes in plant morphology, physiological and biochemical metabolism, and molecular mechanisms. When subjected to drought stress, the plant morphology is firstly affected, and drought symptoms comprise leaf wilting, premature senility yellowing and withering; dysplasia and the like. The physiological and biochemical metabolic levels of plants can also be affected, including photosynthesis weakening, respiratory depression, destruction of cellular structures, oxidative damage, metabolic disorders, and the like.

Plants possess advanced drought tolerance mechanisms to cope with drought stress. When the plant is in early stage of drought stress or has low drought degree, the threshold value of plant resistance is not reached, the plant is mainly resistant to stress, and corresponding physiological activities comprise root elongation, osmotic stress alleviation, hormone regulation and ROS protection; in terms of molecular mechanisms, more variation is focused on: (1) a signal transduction pathway; (2) expression of drought-resistance related genes; (3) Ca (Ca) ²⁺ A signal system; (4) a transcription factor; (5) epigenetic changes. When the later period of drought stress or when drought is severe, the upper limit of plant resistance is exceeded, plants mainly adopt a strategy of drought escape, and early flowering and early fruiting are shown, but the yield is generally reduced.

Drought resistance is a highly complex, compact process that is tightly regulated at multiple levels, including morphological adaptation, physiological adaptation (osmotic pressure regulation, ion and pH balance, antioxidant effects, endogenous hormonal responses, hormonal responses) and molecular regulation (signal transduction, participation of transcription factors, expression of drought-resistant genes and epigenetic regulation). It has been pointed out by the learner that thousands of genes and transcription factors are differentially expressed along with drought stress, and these genes are up-regulated or down-regulated in plants to participate in different signal transduction pathways and networks, and these regulatory networks are interrelated, and multiple cross-talk pathways are in motion to react and regulate drought stress signals of various internal and external environments. In summary, plant drought tolerance is a multi-level regulatory process whose mechanism is well studied in model plants such as Arabidopsis thaliana, which is rarely reported in the case of outcrossing tetraploid crops such as alfalfa.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide stress-resistance related protein MsWRKY2G, and a coding gene and application thereof, wherein MsWRKY2G is a transcription factor, the expression quantity of the MsWRKY2G is increased under drought conditions, and the MsWRKY2G can improve drought resistance of plants through verification.

The invention is realized in the following way:

in a first aspect, the invention provides alfalfa MsWRKY2G protein, the amino acid sequence of which is shown as SEQ ID NO. 2.

In a second aspect, the present invention provides a nucleic acid molecule encoding the alfalfa MsWRKY2G protein described above.

In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the alfalfa MsWRKY2G protein described above is shown in SEQ ID No. 1.

In the present invention, the inventors cloned the MsWRKY2G gene from alfalfa (Medicago sativa l.) of the genus Medicago of the family leguminosae, encoded a protein consisting of 380 amino acids, and expressed it in the nucleus. MsWRKY2G protein is a transcription factor containing a WRKY conserved domain (WRKYGQK) and contains C ₂ -H ₂ The zinc finger structure is divided into groups ii c in the WRKY family.

In a third aspect, the invention also provides a recombinant vector comprising the nucleic acid molecule described above.

In a fourth aspect, the invention also provides a recombinant bacterium, which contains the recombinant vector.

In a fifth aspect, the invention also provides application of the alfalfa MsWRKY2G protein and the coding gene thereof in improving the stress resistance of plants.

In some embodiments, the stress resistance is drought resistance.

In the present invention, drought resistance is the ability to resist water loss stress, wherein water loss stress refers to a natural stress identical or equivalent to simulated stress at a final concentration of 300-500mM mannitol.

In some embodiments, the plants include arabidopsis thaliana, medicago truncatula, and medicago sativa.

The WRKY transcription factor is taken as one of the largest transcription factor families of plants, can regulate the growth and development of the plants, and also can participate in the process of the plants for coping with various biotic stresses and abiotic stresses (such as salt, drought and low temperature). In transcriptome data of drought stress alfalfa, the inventors found that the expression of the MsWRKY2G gene was induced by both salt and drought stress, and that its promoter region contained a large number of stress-related acting elements. Thus, it is speculated that the MsWRKY2G gene may regulate drought tolerance of alfalfa. Further, the inventor utilizes arabidopsis thaliana, medicago truncatula and medicago sativa to carry out functional analysis and verification on the MsWRKY2G gene, and the result shows that under the induction of NaCl and mannitol, the protein coded by the MsWRKY2G gene can improve the drought resistance of plants.

In a sixth aspect, the present invention also provides a method for improving stress tolerance of a plant, comprising introducing the above nucleotide molecule into a genome of a target plant to obtain a transgenic plant seed or a regenerated explant; artificial cultivation is carried out on the transgenic plant seeds or the explants or the transgenic plant seeds are naturally grown; wherein, the stress resistance is drought stress; plants include Arabidopsis thaliana, tribulus medicago sativa, and Medicago sativa.

In some embodiments, the nucleic acid molecules described above are introduced into a plant by a recombinant expression vector.

In some embodiments, the recombinant expression vector is obtained by inserting the above-described nucleic acid molecule into the recombination site of expression vector pBI121 by homologous recombination.

In a seventh aspect, the present invention also provides a plant breeding method comprising: increasing the content and/or activity of the alfalfa MsWRKY2G protein in the plant, thereby enhancing the stress resistance of the plant;

wherein stress resistance is drought stress; plants include Arabidopsis thaliana, tribulus medicago sativa, and Medicago sativa.

The invention has the following beneficial effects:

firstly cloning in alfalfa to obtain an MsWRKY2G gene, wherein the expression quantity of the MsWRKY2G gene is increased under drought conditions, and the encoded protein is positioned to a cell nucleus; secondly, the MsWRKY2G gene can improve the drought resistance of the alfalfa on the premise of not changing the yield and quality of the alfalfa, and is beneficial to cultivating new varieties of the alfalfa with strong drought resistance and expanding the planting scale of the alfalfa. The invention not only provides a research thought for researching drought-resistant genes on a molecular level, but also provides a theoretical basis for improving biomass and quality of pasture crops under drought stress.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is the basic information of the gene MsWRKY 2G;

FIG. 2 is a graph showing the real-time fluorescent quantitative expression results of MsWRKY2G gene induced by stress;

FIG. 3 is a graph showing subcellular localization results of MsWRKY2G in tobacco leaves;

FIG. 4 is a molecular assay of a gene of interest in transgenic Arabidopsis;

FIG. 5 plate mannitol stress assay of transgenic and wild type Arabidopsis;

FIG. 6 is a graph of the drought resistance comparison results of transgenic and wild type Arabidopsis;

FIG. 7 shows the molecular detection of the gene of interest in transgenic alfalfa;

FIG. 8 is a graph showing the comparison of drought resistance of transgenic, wild-type and mutant alfalfa;

FIG. 9 is a physiological index determination after drought stress of transgenic, wild-type, mutant alfalfa;

FIG. 10 is a molecular assay of a gene of interest in transgenic alfalfa;

FIG. 11 is a graph showing the comparison of drought resistance of transgenic and wild alfalfa.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

In this example, the MsWRKY2G gene was cloned and sequenced, and the plant material used in this example was alfalfa number 1 in alfalfa germplasm. The specific operation comprises the following steps:

healthy and full seeds are selected and placed on a culture dish with moist filter paper, and the seeds are placed at the temperature of 4 ℃ for vernalization treatment for 3 days, so that the growth vigor of seedlings after the seeds germinate is consistent. After the vernalization for 3d, the seeds were transferred to a climatic chamber (16/8 h light to dark ratio, 22 ℃) for germination.

And culturing for 3 days, selecting alfalfa seedlings with consistent growth vigor, transferring into 1/2MS nutrient solution (pH=5.8), and performing water culture, wherein the nutrient solution is changed every two days. Plants were placed in a climatic incubator and the growth environment was: photoperiod 16/8h, day and night temperature 25/20 deg.C and relative humidity 60-70%.

After 10d of hydroponics, mannitol was added to the 1/2MS nutrient solution to a final concentration of 400mM, and 6 treatment time points were set at 0, 1,3,6, 12 and 24h. Rapidly taking aerial parts and underground parts of plants respectively, placing into a freezing tube, quick freezing with liquid nitrogen, and storing in a refrigerator at-80deg.C, and storing at-80deg.C.

100mg of plant sample was taken, total RNA in the sample was extracted by Trizo1 method, and genomic DNA was removed by DNaseI. The integrity and purity of the RNA samples were accurately checked by 2100Bioanalyser (Agilent) and ND-2000 (NanoDrop Technologies) methods, respectively, to ensure that the samples were acceptable for use (OD _260/280 ＝1.8～2.2，OD _260/230 ≥2.0，RIN≥6.5，28S:18S≥1.0，>2 μg). The mRNA is then reverse transcribed into cDNA using a cDNA synthesis reverse transcription kit. The primer pairs are MsWRKY2G-F and MsWRKY2G-R, and specific information is shown in Table 1.

TABLE 1 primer information

The total volume of the PCR amplification system was 50. Mu.L, which includes: primer MsWRKY 2G-F1. Mu.L, primer MsWRKY 2G-R1. Mu.L, primeSTAR HSDNA Polymerase (2.5U/. Mu.L) 0.5. Mu.L, dNTP mix (2.5 mM each) 4. Mu.L, 5 XPrimeSTAR Buffer (Mg) ²⁺ Plus) 10 mu L, cDNA mu L and ddH ₂ O 31.5μL。

PCR reaction conditions: first round: pre-denaturation at 98 ℃ for 10s; a second wheel: denaturation at 94℃for 30s, annealing at 58℃for 5s, extension at 72℃for 69s, the second cycle being 35 times; the reaction was terminated at 16℃and completed.

Electrophoresis was performed using 1.5% agarose gel, the length of the target gene was 1140bp, the DNA fragment was recovered using a DNA recovery kit, and the quality of the recovered DNA fragment was detected using 2100Bioanalyser (Agilent).

Then using DNA seamless cloning technique [ ]Ultra One-Step cloning Kit) was ligated into pBI121 expression vector, bamH I and Sac I were selected for cleavage site, and the ligation product was transformed into E.coli strain DH 5. Alpha. After the transformation is completed, the bacterial liquid is uniformly coatedPlacing the mixture on an LB plate (containing kanamycin), placing the mixture into a constant temperature incubator, setting the temperature to be 37 ℃, and culturing the mixture upside down for 12 to 16 hours. Then, the single clone grown on the resistance plate was selected and subjected to colony PCR positive identification (using the primer combination 35S-F and MsWRKY2G-R1, specific information is shown in Table 2).

TABLE 2 primer combination information

Positive monoclonal plaques were placed in LB liquid medium containing Kan resistance and incubated overnight at 37℃in a shaker at 200 rpm. The bacterial liquid was sent to Beijing qingke biotechnology Co.Ltd for sequencing. Sequencing results show that the colony has the nucleotide sequence shown in SEQ ID NO.1, the colony is named as MsWRKY2G gene, the protein coded by the gene is named as MsWRKY2G protein, and the protein sequence is shown as SEQ ID NO. 2.

MsWRKY2G contains a WRKY conserved domain (WRKYGQK) and thus contains C ₂ -H ₂ The zinc finger structure, divided into group ii C in the WRKY family, encodes a protein consisting of 380 amino acids (fig. 1A-C). The MsWRKY2G protein was aligned on GeneBank (FIG. 1D), and had the highest homology to MTR_2g033820 of alfalfa, and homologous genes of this gene in these species all belong to the WRKY transcription factor family.

Example 2

The present example is a real-time fluorescent quantitative PCR analysis of the expression characteristics of MsWRKY2G, comprising the steps of:

alfalfa No.1 seed germination and hydroponic method is the same as in example 1. 12-day-old alfalfa seedlings were subjected to the following stress treatments:

drought treatment: mannitol was added to the 1/2MS hydroponic nutrient solution to a final concentration of 500mM and treated for 0, 1,3,6, 12 and 24 hours, respectively. The overground and underground parts are sampled respectively, and the liquid nitrogen is quickly frozen and stored in a refrigerator at the temperature of minus 80 ℃ for standby.

And (3) NaCl treatment: naCl was added to the 1/2MS hydroponic nutrient solution to a final concentration of 150mM, and treated for 0, 1,3,6, 12 and 24 hours, respectively. The overground and underground parts are sampled respectively, and the liquid nitrogen is quickly frozen and stored in a refrigerator at the temperature of minus 80 ℃ for standby.

ABA (abscisic acid) treatment: ABA was added to the 1/2MS hydroponic nutrient solution to a final concentration of 80 μm and treated for 0, 1,3,6, 12 and 24h, respectively. The overground and underground parts are sampled respectively, and the liquid nitrogen is quickly frozen and stored in a refrigerator at the temperature of minus 80 ℃ for standby.

Sample RNA extraction and reverse transcription cDNA methods were as in example 1. The cDNA was diluted to 50 ng/. Mu.L. The expression level of MsWRKY2G gene was detected by qRTMsWRKY2G-F and qRTMsWRKY2G-R, and MsAbin-F and MsAbin-R were used as internal references, and specific primer information is shown in Table 3.

TABLE 3 primer information

The total volume of the qRT-PCR detection system was 20. Mu.l, which included: primer qRTMsWRKY 2G-F0.5. Mu.L, primer qRTMsWRKY 2G-R0.5. Mu.L, 2xSG Fast qPCR Master Mix 10. Mu.L, DNF buffer 2. Mu. L, cDNA 2. Mu.L and ddH ₂ O 5μL。

qRT-PCR reaction conditions: first round: pre-denaturation at 95℃for 10min; a second wheel: denaturation at 95℃for 15s, annealing at 60℃for 5s, and a second cycle of 40 times; the reaction was terminated at 16℃and completed. Each reaction was performed in 3 replicates.

The instrument was ABI7500 real-time fluorescence quantitative PCR, and the mapping software was Excel 2019.

2 ^-△△Ct The method is used to calculate the relative expression:

△△Ct＝(Ct _{target gene} -Ct _{Reference gene} ) _Time _x -(Ct _{Target gene} -Ct _{Reference gene} ) _Time ₀ ；

Time x represents the stress processing Time, i.e., x represents 1,3,6, 12 or 24h. Time 0 indicates that no stress treatment was performed (control group).

As a result, as shown in FIG. 2, the MsWRKY2G gene was expressed in the roots, stems, leaves and flowers of alfalfa, while the expression level in flowers was highest, and next to that in stems, the expression level of MsWRKY2G in roots was lowest. Under abiotic stress, the expression of the MsWRKY2G gene was induced to varying degrees by drought and salt stress in both the aboveground and underground parts of alfalfa (FIGS. 2A-K).

Example 3

This example is an MsWRKY2G subcellular localization assay comprising the steps of:

1. material preparation

The plant material is Nicotiana benthamiana. Healthy and full Nicotiana benthamiana seeds are soaked in distilled water for 2 hours and then sown in soil, water is poured once every week, and tobacco after 6-7 weeks can be used for subcellular localization.

2. Construction of subcellular vectors

The subcellular localization vector is pBI121-eGFP. The full length of CDS of MsWRKY2G was amplified using primers MsWRKY2G-eGFP-F and MsWRKY2G-eGFP-R containing BamHI and SacI cleavage sites using the alfalfa cDNA of example 1 as a template, and specific primer information is shown in Table 4.

TABLE 4 primer information

The total volume of the PCR amplification system was 50. Mu.L, which includes: primer MsWRKY 2G-eGFP-F1. Mu. L, msWRKY 2G-eGFP-R1. Mu.L, primeSTAR HSDNA Polymerase (2.5U/. Mu.L) 0.5. Mu.L, dNTP mix (2.5 mM each) 4. Mu.L, 5 XPrimeSTAR Buffer (Mg) ²⁺ Plus) 10 mu L, cDNA mu L and ddH ₂ O 31.5μL。

PCR reaction conditions: first round: pre-denaturation at 98 ℃ for 10s; a second wheel: denaturation at 94℃for 30s, annealing at 66℃for 5s, elongation at 72℃for 70s, the second cycle being 35 times; the reaction was terminated at 16℃and completed.

Electrophoresis was performed using 1.5% agarose gel, the length of the target gene was 1158bp, the DNA fragment was recovered using a DNA recovery kit (full gold), and the quality of the recovered DNA fragment was detected using 2100Bioanalyser (Agilent).

Cutting the recovered DNA fragment by using restriction enzymes BamHI and SacI, and recovering the digested PCR product again, wherein the size of the digested product is 1140bp; likewise, the pBI121-eGFP expression vector was digested with the restriction enzymes BamHI and SacI, and the digested vector fragment was recovered, and the size of the digested product was 1.4kb.

The target gene-digested and purified product and the pBI121-GFP expression vector-digested and purified product were ligated overnight at 4℃with DNA ligase (TakaRa, cat# 6022). The ligation product was transferred into E.coli DH 5. Alpha. By a heat shock method, cultured overnight at 37℃and identified and sequenced by PCR to obtain the fusion vector pBI121-eGFP-MsWRKY2G. And respectively converting the fusion vector and the empty plasmid into agrobacterium EHA105 by an electric shock conversion method to obtain recombinant agrobacterium EHA105/pBI121-eGFP-MsWRKY2G, mixing bacterial liquid with 50% glycerol according to a volume ratio of 1:1, and preserving at-80 ℃.

3. Preparation of Agrobacterium infection liquid

Small shake: recombinant Agrobacterium pBI121-eGFP-MsWRKY2G frozen at-80℃was placed on ice, 50ul of Agrobacterium was pipetted into 10mL LB liquid medium (1:500; 50. Mu.g/mL Kan+20. Mu.g/mL Rif), 180rpm, and shake cultured at 28-30℃for 24h. Rif is the agrobacterium strain EHA105 carrying resistance and Kan is the expression vector carrying resistance.

Large shaking: 100ul of the overnight cultured Agrobacterium were transferred to 100mL of LB liquid medium (1:1000; 50. Mu.g/mL Kan+20. Mu.g/mL Rif). Shake culturing at 180rpm and 28-30 deg.C for 15 hr, and detecting the bacterial liquid OD ₆₀₀ Detecting the value of the (D) of the bacterial liquid grown to the late exponential phase OD ₆₀₀ ＝0.6-0.8。

Collecting bacterial liquid of 2400g and 15min, removing supernatant, re-suspending bacterial body with re-suspension (which can be re-suspended with half volume of bacterial liquid), and final OD ₆₀₀ 0.4 (0.1-0.8 as needed, not exceeding 1, too high a concentration would damage the blade). Standing the heavy suspension at 28-30 ℃ for 1-3h (not more than 6 h), and carrying out heavy suspension on pBI 121-eGFP-empty and Marker-NLS, pBI121-eGFP-WRKY2G and Marker-NLS according to the following ratio of 1:2, and injecting tobacco after mixing according to the proportion.

The preparation method of the heavy suspension comprises the following steps: 10mM MES+10mM MgCl ₂ +100uM AS, mixAfter being evenly preserved at 4 ℃, the medicine is prepared for use.

4. Injection method for transforming tobacco leaves

And (3) selecting tobacco leaf plants with good growth state and strong growth state for 6-7 weeks to carry out agrobacterium infection. Watering thoroughly before transformation, and controlling watering after transformation.

Sucking the settled heavy suspension by a 1mL syringe (needle removed); the vein is avoided, after a small hole is formed in the blade by using the injector needle, the left hand props against the front surface of the blade, the injection pipe orifice is lightly pressed on the back surface of the blade by the right hand, the thumb of the right hand is slowly and evenly pushed and pressed with force, and bacterial liquid is observed to slowly move under the epidermis of the blade until the whole blade is infected. The injected tobacco should be kept moist, covered with a black plastic bag (24 h), and returned to the culture chamber.

The best time is observed 2.5-3 days after injection, preferably not more than 5 days, and the affected area is cut into small pieces (1 cm) ² Left and right), the glass slide was placed on a glass slide with water dropped thereon (with the lower surface placed thereon) after being placed on a glass slide containing 0mM, 50mM, 150mM, 300mM mannitol for 0h, 1h, 3h, 6h, and the cover glass was gently covered to prevent the generation of air bubbles. Firstly observing under a fluorescence microscope, photographing by using a confocal microscope if the expression is expressed, observing under x20 and x40 in sequence, and finally photographing by using a x40 focal length, wherein tobacco transformed into a cell nucleus Maker (mCherry, RFP) is used as a marker. The excitation wavelengths of eGFP and Mcherry were 484nm and 587nm, respectively.

As a result, as shown in FIG. 3, the MsWRKY2G gene was localized in the nucleus and was not changed by drought stress.

Example 4

The embodiment is the application of MsWRKY2G in improving the stress resistance of Arabidopsis thaliana

Noteworthy are: the invention uses Arabidopsis thaliana as a model plant to carry out functional analysis and verification on MsWRKY2G genes, and the conclusion can be considered to be equivalent to the molecular mechanism of MsWRKY2G in alfalfa for regulating and controlling drought response of plants.

The method comprises the following steps:

1. transformation of MsWRKY2G Arabidopsis

1. Recombinant Agrobacterium obtaining

The pBI121-MsWRKY2G overexpression vector obtained in example 1 is transformed into agrobacterium EHA105 to obtain recombinant agrobacterium EHA105/pBI121-MsWRKY2G, and the bacterial liquid and 50% glycerol are mixed according to the volume ratio of 1:1 and stored at-80 ℃.

2. Obtaining the MsWRKY2G arabidopsis thaliana

Recombinant Agrobacterium pBI121-MsWRKY2G frozen at-80℃was placed on ice, and 50ul of Agrobacterium was extracted and cultured in 10mL LB liquid medium (50. Mu.g/mL Kan+20. Mu.g/mL Rif), 180rpm, and shaking culture at 28-30℃for 24 hours. Rif is the agrobacterium strain EHA105 carrying resistance and Kan is the expression vector carrying resistance.

1mL of Agrobacterium parvum was transferred to 100mL of LB liquid medium (50. Mu.g/mL Kan+20. Mu.g/mL Rif). Shake culturing at 180rpm and 28-30 deg.C for 20-24 hr, and detecting the bacterial liquid OD of overnight culture ₆₀₀ Detecting the value of the (D) of the bacterial liquid grown to the late exponential phase OD ₆₀₀ =1.2-2.0. Collecting part of bacterial liquid (2400 g,15 min), removing supernatant, and re-suspending to OD of flower soaking buffer solution with 5% sucrose ₆₀₀ ＝0.8。

Soaking an arabidopsis thaliana (Col-0) inflorescence into an invasion dye solution, and infecting for 3-5 seconds; after the soaking, the flowerpot is taken out, covered with a black plastic bag (24 h), and continuously cultivated in a greenhouse. Infection was 2 nd after 1 week.

T1 generation seeds were harvested, positive plants were screened for resistance using kanamycin (Kan, 70 mg/mL), and passaged until T3 generation yielded homozygous lines.

The T2 generation represents the seed generated by T1 generation selfing and the plant grown by the seed, and the T3 generation represents the seed generated by T2 generation selfing and the plant grown by the seed.

The DNA of leaf of the T3 generation regenerated plant is used as a template, and chimeric primer combination 35S-F (SEQ ID NO. 5) and MsWRKY2G-R1 (SEQ ID NO. 6) are used for PCR detection to verify whether the plant is a positive plant.

The total volume of the PCR detection system was 25. Mu.L, which includes: upstream primer 35S-F1. Mu.L, downstream primer MsWRKY2G-R1 1. Mu.L, 2X Rapid Taq Master Mix 12.5. Mu. L, cDNA 2. Mu.L and ddH ₂ O 8.5μL。

PCR reaction conditions: first round: pre-denaturation at 95℃for 3min; a second wheel: denaturation at 95℃for 15s, annealing at 58℃for 15s, extension at 72℃for 17s, the second cycle being 35 times; the reaction was terminated at 16℃and completed.

Electrophoresis was performed using a 1.5% agarose gel, and as shown in FIG. 4A, the T3 generation 12 homozygous lines amplified a single band at 1131 bp.

Extracting RNA of the whole plant of the T3 generation regeneration plant, reversely transcribing the RNA into cDNA as a qRT-PCR template, detecting the expression quantity of the MsWRKY2G gene by using a target gene part fragment primer pair qRTMsWRKY2G-F and qRTMsWRKY2G-R, and carrying out qRT-PCR amplification by using AtACTIN-F and AtACTIN-R as internal references, wherein the primer information of the AtACTIN is shown in a table 5.

Table 5 primer information for reference AtACTIN

The total volume of the qRT-PCR detection system was 20. Mu.L, which includes: primer qRTMsWRKY 2G-F0.5. Mu.L, primer qRTMsWRKY 2G-R0.5. Mu.L, 2xSG Fast qPCR Master Mix 10. Mu.L, DNF buffer 2. Mu. L, cDNA 2. Mu.L and ddH ₂ O 5μL。

As a result, as shown in FIGS. 4A-B, msWRKY2G was not expressed in the wild-type strain, and was expressed to a different extent in the homozygous strain. 3 homozygous lines with high expression were selected for subsequent physiological analysis.

2. Drought resistance evaluation of transgenic Arabidopsis thaliana

1. Effects of drought stress on germination Rate

After subjecting each 49 seeds of 3 representative strains OE2, OE7, OE9 and wild-type Col-0 of T3-generation MsWRKY2G Arabidopsis to sterilization treatment, uniformly dispensing with 10. Mu.L of a gun head on MS medium and MS medium containing 100, 200, 300 or 400mM mannitol, sealing with a sealing film, treating at a low temperature of 4 ℃ for 72 hours, transferring into an incubator at 22 ℃ for 16/8 hours and 60% relative humidity, culturing for 7 days, counting germination rate every day, setting three biological replicates for each treatment, taking an average value of the results, and calculating standard deviation.

The results are shown in FIGS. 5A-C, where the germination rates and rates of wild-type and transgenic lines were substantially identical on MS plates; on a 300mmo1/L mannitol plate, the germination rate and the green seedling rate of the strain OE9 of the MsWRKY 2G-to-Arabidopsis are both obviously larger than those of the wild type strain OE 9.

2. Effects of drought stress on root length

After 100 healthy and full seeds of T3-generation MsWRKY2G Arabidopsis thaliana are disinfected, uniformly and respectively dotted in an MS culture medium by using a10 mu L gun head, sealing by using a sealing film, performing low-temperature treatment at 4 ℃ for 72 hours, transferring the seeds into a 22 ℃ and 16/8h light/dark 60% relative humidity incubator for 7 days, selecting 3 transgenic lines with consistent main root length and wild type Col-0 seedlings, respectively transferring the seedlings onto the MS culture medium and the MS culture medium containing 300mM mantiol, counting root lengths of the lines after 12 days of culture, setting three biological repetitions for each treatment, taking an average value of the results, and calculating standard deviation.

As a result, as shown in FIG. 5D, the root lengths of the wild-type and transgenic lines were substantially identical on the MS plates; on a 300mmo1/L mannitol plate, the root length of the MsWRKY2G transgenic Arabidopsis line is obviously larger than that of the wild type.

3. Influence of soil drought stress on plants

After 100 healthy and full seeds of T3 generation MsWRKY2G Arabidopsis thaliana are disinfected, uniformly and respectively dotted in MS culture medium by using 10 mu L gun heads, sealing by sealing films, performing low-temperature treatment at 4 ℃ for 72 hours, transferring the seeds into an incubator with the temperature of 22 ℃, the light/dark for 16/8 hours and the relative humidity of 60 percent for culturing for 14 days, transferring the seedlings into small square basins with consistent soil weight, normally culturing 9 seedlings in each basin for 14 days, watering to saturation, cutting off water, performing drought stress treatment for 24 days until the wild plants wilt, then starting rehydration for 2 days, observing plant phenotypes, counting survival rate, photographing, and detecting physiological indexes before and after drought treatment.

The results are shown in FIGS. 6A-D, the transgenic lines and wild-type growth conditions were substantially identical prior to drought; after drought, the transgenic strain grows better than the wild strain, and the wilting degree of the wild strain is more serious; after rehydration, the survival rate of the transgenic lines was significantly higher than that of the wild type (fig. 6A-B), and the in vitro leaf loss rate of the transgenic lines was also lower than that of the WT plants (fig. 6C). Therefore, the plants transformed with the MsWRKY2G gene can show stronger drought resistance by controlling water loss.

Before drought treatment, there was no obvious difference in physiological index between wild type and transgenic lines; after 21d drought treatment, both the ion leakage rate (EL) and malondialdehyde content of the transgenic and wild type plants increased significantly, but the membrane damage was smaller for the transgenic plants, which had significantly lower ion leakage levels and malondialdehyde content than the wild type plants (P < 0.05). To avoid oxidative damage, plants maintain redox homeostasis by activating the antioxidant enzyme defense system. The assay also measures the antioxidant enzyme activity of wild-type and transgenic lines before and after drought treatment. The results showed that the enzyme activities of the transgenic plants POD and CAT were significantly increased under drought stress conditions compared to the control, and the POD and CAT enzyme activities of the wild-type plants were lower than those of the transgenic plants (P < 0.05) (fig. 6D), indicating that the transgenic plants were more effective in alleviating oxidative damage under drought stress conditions.

Example 5

The embodiment is the application of MsWRKY2G in improving the stress resistance of medicago truncatula

Noteworthy are: the invention uses the alfalfa as leguminous mode plant to carry out functional analysis and verification on the MsWRKY2G gene, and the conclusion can be considered to be equivalent to the molecular mechanism of MsWRKY2G regulation plant response drought in the alfalfa.

1. Alfalfa transformed into MsWRKY2G caltrop

1. Recombinant Agrobacterium obtaining

Recombinant Agrobacterium of example 4 was used.

2. Obtaining the medicago sativa with MsWRKY2G

The ecological type R108 medicago sativa is used as a test material, the leaves of the R108 aseptic seedling are used as genetic transformation explants, and the overexpression vector (pBI 121-MtWRKY 2G) after sequencing verification is transformed into R108 by an agrobacterium infection method to obtain the transgenic medicago sativa regenerated plant. The specific genetic transformation method is as follows:

1mL of Agrobacterium parvum was transferred to 100mL of LB liquid medium (50. Mu.g/mL Kan+20. Mu.g/mL Rif). Shake culturing at 180rpm and 28-30 deg.C for 20-24 hr, and detecting the bacterial liquid OD of overnight culture ₆₀₀ Detecting the value of the (D) of the bacterial liquid grown to the late exponential phase OD ₆₀₀ ＝0.6-0.8。

Precooling (4 ℃) by a centrifuge, centrifuging the bacterial liquid for 15min by using a centrifugal force of 2,400g, discarding the supernatant, only reserving bacterial cells, and re-suspending the bacterial cells by using SH3A liquid culture medium, so that the OD (lambda=600nm) value of the finally re-suspended bacterial liquid is about 0.3, and taking the bacterial liquid as an invader liquid.

In an ultra-clean workbench, 6-8 fresh alfalfa complex leaves are collected, placed into an infection liquid, and then vacuumized in a dryer for 10min.

In the ultra clean bench, the infested solution attached to the blade was blotted dry using sterile filter paper.

The leaves were transferred to SH3A co-culture medium, wrapped with tinfoil, and dark-cultured at 24℃for 2d until a small number of colonies appeared at the edges of the leaves.

Sequentially culturing the alfalfa leaves of the caltrops in SH3A selective medium (about 4 weeks) until the calli are enlarged; SH9 medium (about 4-6 weeks of culture) to root; culture conditions: the temperature is 24 ℃, and the photoperiod is 16h illumination/8 h darkness; the culture medium was changed every 15d during the culture.

After the complete root, stem and leaf tissue culture seedling grows, transferring the tissue culture seedling to a 1/2MS culture medium for 20d, and transplanting the seedling to pure vermiculite for culture. The seedlings are transplanted from the tissue culture bottle to the soil for 7 days, and care needs to be taken to keep moisture for regenerated plants, so that the survival rate is improved. Leaf DNA is extracted as an amplification template, and chimeric primers consisting of specific sequences on 35S-F and MtWRKY2G genes are used for PCR detection of transgenic alfalfa regenerated plants. After positive regeneration plants are determined, leaf RNA is extracted and reversely transcribed into cDNA, and qRT-PCR detection is carried out on the expression level of MtWRKY2G genes in the over-expression plants.

Furthermore, two mutant lines of MtWRKY2G were obtained in the alfalfa Tnt mutant pool of tribulus terrestris: NF8978 and NF9753.

As a result, as shown in FIGS. 7A-M, msWRKY2G was not expressed in the wild-type strain, and was expressed to a different extent in the transgenic strain. 3 homozygous lines with high expression were selected for subsequent physiological analysis.

2. Drought resistance evaluation of transgenic medicago sativa

1. Phenotype analysis of MtWRKY2G over-expression and mutant alfalfa under drought stress

Selecting full alfalfa seeds (WT, OE12, OE19, OE21, mtWRKY2g-1 and mtWRKY2 g-2) with consistent size, lightly polishing with sand paper, and spreading the seeds on distilled water soaked filter paper after the seed coats are tiny scratched. After the seeds are swelled, the seeds are placed in a 4 ℃ low-temperature light-shielding condition for vernalization for 48 hours, and the seeds are transferred to an illumination incubator for germination after finishing. Transplanting the seedlings after 10d germination to pure vermiculite, and placing the plants in a condition that the illumination intensity is 140 mu mol m ^-2 s ^-1 Culturing in a dark incubator at 25deg.C and humidity of 70% with a photoperiod of 16/8 hr. After the alfalfa seedlings grew for 30d, watering was stopped for drought treatment, each treatment was repeated 3 times, and their phenotypes were observed and survival was counted.

2. Determination of physiological index of MtWRKY2G over-expressed medicago sativa under drought stress

The alfalfa plants were planted in the soil, seedlings grown in the soil for 14d were selected for drought stress for 20d, 0.1g of fresh leaves per genotype were collected as measurement samples, and 3 biological replicates were set per treatment. And then performing SOD, POD and CAT activity determination tests by using an antioxidant enzyme activity kit of Suzhou Ming biotechnology Co Ltd, wherein the specific steps are operated according to the kit instruction.

The results are shown in FIG. 8, the transgenic lines and wild-type growth conditions were substantially identical prior to drought; after drought, the transgenic strain grows better than the wild strain, and the wilting degree of the wild strain is more serious; after rehydration, the survival rate of the transgenic lines was significantly higher than that of the wild type.

Under normal conditions, there was no significant difference in antioxidant enzyme activity between wild type and overexpressed alfalfa; and after 30d drought treatment, the malondialdehyde content of the transgenic plants and wild plants is obviously increased, but the malondialdehyde content of the WT plants is obviously higher than that of the transgenic plants. In addition, the activities of the oxidase (POD), catalase (CAT) and superoxide dismutase (SOD) enzymes were all significantly higher than wild type (P < 0.05) in MtWRKY2G overexpressing lines after drought treatment (fig. 9). These results indicate that overexpression of the MtWRKY2G gene in medicago truncatula can effectively alleviate oxidative damage under drought stress.

Example 6

The embodiment is the application of MsWRKY2G in improving the stress resistance of alfalfa

1. Alfalfa transformed with MsWRKY2G

1. Recombinant Agrobacterium obtaining

Recombinant Agrobacterium of example 4 was used.

2. Obtaining the alfalfa transformed by MsWRKY2G

The SY4D germplasm alfalfa leaf is adopted as an explant, and a plant expression vector is transformed into alfalfa by an agrobacterium-mediated method to obtain a transgenic regenerated plant.

As a result, as shown in FIGS. 10A-H, msWRKY2G was not expressed in the wild-type strain, and was expressed to a different extent in the transgenic alfalfa strain. 3 high-expression transgenic lines were selected for subsequent physiological analysis.

2. Drought resistance evaluation of transgenic alfalfa

1. Soil drought stress evaluation test

3 genotypes of Arabidopsis thaliana were germinated on 1/2MS medium, and 12-day-old Arabidopsis thaliana seedlings after germination were selected and transplanted to planting soil (turfy soil: vermiculite=1:3) with consistent vigor. Arabidopsis seedlings grown in soil for about 20d were transplanted, a water-rich treatment was set as a control group, and a drought stress treatment group was set, each treatment was subjected to 3 biological replicates, the phenotype of the treatment group and the control group was observed and photographed, and survival was counted.

Determination of drought stress physiological index of MsWRKY2G over-expressed Arabidopsis thaliana

Transgenic and wild arabidopsis seedlings which grow for 20 days in soil and have consistent growth vigor are selected, and normal conditions and drought stress treatment are set. After 16d of drought stress, 0.1g of fresh leaves per genotype was collected as a measurement sample, and 3 biological replicates were set per treatment. Physiological changes in plants under drought stress and normal conditions, including malondialdehyde levels, ion leakage rates, and enzyme activities of CAT and POD were determined.

The malondialdehyde content, CAT and POD enzyme activities were determined using a kit (available from Suzhou Ming Biotechnology Co., ltd.) and the specific method was performed according to the kit instructions.

The leaf ion leakage rate (electrolyte leakage, EL) was measured as follows: the normal conditions and the ex vivo leaves after drought stress were placed in centrifuge tubes containing 15 ml deionized water, respectively. The leaves were cooked in a water bath at 100 ℃ for 20min, after cooling to room temperature, the conductivity before boiling (Ci) and after cooling (Cmax) were measured, respectively, and the ion leakage rate was calculated according to the formula EL (%) = (Ci/Cmax) ×100.

The method for detecting the water loss rate of the in-vitro blade comprises the following steps: arabidopsis plants grown under conditions of sufficient moisture and 3 weeks old were selected for testing, rosette leaves were collected and immediately weighed, and at least 30 rosette leaves per line were taken. At room temperature, weighing is carried out once every one hour, weighing is carried out continuously for 8 hours, each strain is set for at least 3 times of repetition, and the water loss of the leaf per unit time is calculated according to the initial weight of the leaf.

Drought resistance evaluation of MsWRKY2G overexpressed alfalfa

In order to obtain alfalfa seedlings with consistent growth vigor, asexual propagation is carried out on wild alfalfa (SY 4D) and over-expressed MsWRKY2G transgenic alfalfa in a cutting mode, after 21D of cutting, seedlings with consistent growth vigor are transplanted into flowerpots with the size of 9 multiplied by 9cm, vermiculite is selected as a growth medium, and 1/2MS nutrient solution is applied regularly at the same time, so that the wild plants and the transgenic plants are ensured to be in the same growth state. After the alfalfa cutting seedlings grow normally for 30d, the alfalfa complex leaves are collected to measure the water loss rate of the in-vitro leaves, and the measuring method is the same as that described above. In addition, wild type plants with consistent growth vigor and 30 days old and MsWRKY2G transgenic alfalfa plants are selected for drought treatment. Photosynthesis metrics (including intercellular carbon dioxide concentration, net photosynthetic rate, stomatal conductance and transpiration rate) were measured before and after drought treatment, respectively, using a photosynthetic apparatus (LI-6400, LI-COR, nebraska, USA), and 5 replicates were set for each strain.

As shown in FIGS. 11A-E, under normal conditions, net photosynthetic rate, stomatal conductance and transpiration rate were significantly higher in MsWRKY2G overexpressed alfalfa than in wild-type, while intercellular CO ₂ The concentration is not obviously different from that of wild alfalfa. After drought treatment 14d, either wild-type or MsWRKY2G overexpressing alfalfa, their net photosynthetic rate, stomatal conductance and transpiration rates were inhibited by drought stress. Wherein the net photosynthetic rate, stomatal conductance and transpiration rate of overexpressed alfalfa are still higher than those of wild-type (P<0.05 CO) between cells ₂ The concentration was significantly different from the wild type. These results indicate that overexpression of MsWRKY2G can increase photosynthetic capacity of alfalfa under drought stress. In addition, the present example also measured the water loss rate of the in vitro leaf of the over-expressed plants and wild alfalfa, and found that the water loss rate of the transgenic alfalfa leaf was significantly lower than that of the WT plant.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An alfalfa MsWRKY2G protein comprising an amino acid sequence as set forth in any one of (1) to (2):

(1) An amino acid sequence shown in SEQ ID NO. 2;

(2) And (3) the protein which is derived from (1) and is related to plant stress resistance through substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO. 2.

2. A nucleic acid molecule encoding the alfalfa MsWRKY2G protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID No. 1.

4. A recombinant vector comprising the nucleic acid molecule of claim 2 or 3.

5. A recombinant bacterium comprising the recombinant vector according to claim 4.

6. The use of alfalfa MsWRKY2G protein of claim 1, its coding gene in improving plant stress-resistance.

7. The use of claim 6, wherein the stress resistance is drought stress.

8. The use according to claim 7, wherein said plants comprise arabidopsis thaliana, medicago truncatula and medicago sativa.

9. A method for improving stress resistance of a plant, comprising introducing the nucleotide molecule of claim 2 or 3 into a genome of a target plant to obtain a transgenic plant seed or a regenerated explant; artificial cultivation of the transgenic plant seeds or explants or natural growth of the transgenic plant seeds;

preferably, the stress resistance is drought stress;

preferably, the plants include arabidopsis thaliana, medicago truncatula and medicago sativa.

10. A plant breeding method, comprising: increasing the content and/or activity of the alfalfa MsWRKY2G protein of claim 1 in a plant, thereby enhancing the stress-resistance of the plant;

preferably, the stress resistance is drought stress;