A Medicago falcata WRKY47 transcription factor, gene and its application in improving plant salt tolerance and / or drought tolerance

Abstract

The present invention provides a Medicago falcata WRKY47 transcription factor, a gene thereof, and the application thereof in improving the salt tolerance and / or drought resistance of plants, belonging to the technical field of bio-agriculture. The present invention provides a Medicago falcata WRKY47 transcription factor, the amino acid sequence of which is as shown in SEQ ID NO: 1. The Medicago falcata WRKY47 transcription factor of the present invention improves the salt tolerance and / or drought resistance of plants through positive regulation. The examples of the present invention show that MfWRKY47 the expression level can respond to the changes in the salt concentration and drought degree in the growth environment, and heterologous expression MfWRKY47 significantly improves the salt tolerance and drought resistance of transgenic plants compared with wild-type plants. The Medicago falcata WRKY47 transcription factor of the present invention enriches the genetic resource library of salt tolerance and / or drought resistance, and has important practical application significance for the breeding of stress-resistant plant lines.

Description

Technical Field

[0001] The present invention belongs to the technical field of bio - agriculture, and particularly relates to a Medicago falcata WRKY47 transcription factor, a gene thereof, and their application in improving plant salt tolerance and / or drought tolerance. Background Art

[0002] Drought and high salinity are abiotic stress factors that severely restrict plant nutrient absorption and growth. Drought stress and salt stress lead to a decline in the quality and yield of many crops, seriously threatening the development of agricultural production. During the process of evolution, plants have acquired a comprehensive signal network regulation through transcription factors, hormones, protein kinases, stress - related proteins, and receptors to adapt to corresponding adverse stress. Among them, transcription factors play a core regulatory role. Therefore, transgenic molecular breeding is an effective method to improve the stress resistance of plants.

[0003] Medicago falcata ( Medicago falcate ) is widely distributed in cold regions and has characteristics such as drought resistance, cold resistance, salt and alkali tolerance, wind and sand resistance, and barren tolerance. Its ecological adaptability and stress resistance are superior to those of Medicago sativa, and it has a rich genetic background, making it an important gene pool for resistance breeding of Medicago crops. However, there are currently few reports on stress - related functional genes of Medicago falcata. When conducting transgenic molecular breeding, the gene resources available for improving plant salt tolerance and drought tolerance are insufficient. Summary of the Invention

[0004] In view of this, the present invention provides a Medicago falcata WRKY47 (abbreviated as MfWRKY47) transcription factor, which can improve plant salt tolerance and / or drought tolerance through positive regulation.

[0005] To achieve the above - mentioned purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a Medicago falcata WRKY47 transcription factor, and its amino acid sequence is as shown in SEQ ID NO: 1.

[0007] The present invention provides a gene encoding the Medicago falcata WRKY47 transcription factor.

[0008] The present invention provides a gene - derived product containing the above - mentioned gene, including an expression cassette containing the gene;

[0009] The expression cassette further contains a promoter, and the nucleotide sequence of the promoter is as shown in SEQ ID NO: 3.

[0010] The present invention provides the application of the Medicago falcata WRKY47 transcription factor, the gene, or the gene - derived product in at least one of the following works:

[0011] 1) Improve the salt tolerance and / or drought tolerance of plants;

[0012] 2) Create salt-tolerant and / or drought-tolerant plant varieties.

[0013] Preferably, the Medicago falcata WRKY47 transcription factor or the gene improves the salt tolerance and / or drought tolerance of plants through positive regulation.

[0014] Preferably, the improvement of the salt tolerance and / or drought tolerance of plants includes changes in at least one of the following indicators: increasing plant biomass, increasing the chlorophyll content of plants, reducing the accumulation of H2O2 in plants and / or the accumulation of O 2- accumulation, increasing the antioxidant enzyme activity of plants and reducing the malondialdehyde content of plants.

[0015] Preferably, the plants include Medicago plants or Arabidopsis plants.

[0016] The present invention provides a method for improving the salt tolerance and / or drought tolerance of plants, which overexpresses the Medicago falcata WRKY47 transcription factor, the gene or the gene-derived product in plants.

[0017] The present invention provides a method for identifying the salt tolerance and / or drought tolerance of plants, which detects the expression level of the Medicago falcata WRKY47 transcription factor or the gene in plants, and judges the salt tolerance and / or drought tolerance of plants according to the expression level:

[0018] The expression level of the Medicago falcata WRKY47 transcription factor or the gene is positively correlated with the salt tolerance and / or drought tolerance of plants.

[0019] Preferably, the reagents for detecting the expression level of the gene include the forward primer shown in SEQ ID NO: 4 and the reverse primer shown in SEQ ID NO: 5.

[0020] The present invention has the following advantages compared with the prior art:

[0021] The present invention provides a Medicago falcata WRKY47 transcription factor, and the amino acid sequence is as shown in SEQ ID NO: 1. The Medicago falcata WRKY47 transcription factor of the present invention improves the salt tolerance and / or drought tolerance of plants through positive regulation. Examples of the present invention show that MfWRKY47 the expression level will respond to changes in salt concentration and drought environment, and heterologous expression MfWRKY47 the salt tolerance and drought tolerance of Arabidopsis plants are significantly improved compared with wild-type plants. The Medicago falcata WRKY47 transcription factor of the present invention enriches the gene resource library of salt tolerance and / or drought tolerance, and has important practical application significance for the breeding of stress-resistant plant lines.

[0022] The present invention provides a method for improving the salt tolerance and / or drought resistance of plants, by transferring the gene or a derivative containing the gene into plants. In the examples of the present invention, MfWRKY47 Arabidopsis thaliana was genetically transformed by the floral dip method, and the heterologous expression MfWRKY47 Arabidopsis thaliana showed significantly improved salt tolerance and / or drought resistance compared with wild-type Arabidopsis thaliana. Description of the Drawings

[0023] Figure 1 It is a hydroponic seedling diagram of Medicago falcata;

[0024] Figure 2 It is Medicago falcata MfWRKY47 Phylogenetic tree analysis diagram;

[0025] Figure 3 It is Medicago falcata MfWRKY47 Tissue expression characteristics analysis diagram;

[0026] Figure 4 It is Medicago falcata MfWRKY47 Analysis result diagram of the expression pattern in response to salt and drought stresses; where A is drought stress; B is salt stress;

[0027] Figure 5 It is Medicago falcata MfWRKY47 Analysis result diagram of the expression pattern driven by the promoter of GUS; where A is CK, B is salt stress, C is drought stress, and D is the GUS activity under drought stress;

[0028] Figure 6 It is the subcellular localization verification result diagram of MfWRKY47 in Medicago falcata, and the scale bar is 20μm;

[0029] Figure 7 It is the transcriptional activation function verification result diagram of MfWRKY47 in Medicago falcata;

[0030] Figure 8 It is the verification result diagram of the binding of MfWRKY47 to the W-box in Medicago falcata;

[0031] Figure 9 It is the verification result diagram of the expression level of the transgenic lines with heterologous expression MfWRKY47 in Arabidopsis thaliana;

[0032] Figure 10 It is the verification result diagram of the salt tolerance and drought resistance of the transgenic lines with heterologous expression MfWRKY47 in Arabidopsis thaliana; where A is the seed germination rate; B is the primary root length; C is the fresh weight of the plant;

[0033] Figure 11 It is the analysis result diagram of the leaf water loss rate of the transgenic lines with heterologous expression MfWRKY47 after drought stress treatment;

[0034] Figure 12 Chlorophyll content measurement results of transgenic lines for heterologous expression MfWRKY47 ;

[0035] Figure 13 Chlorophyll content measurement results of transgenic lines for heterologous expression MfWRKY47 H2O2 content measurement and DAB staining results of transgenic lines for heterologous expression

[0036] Figure 14 Chlorophyll content measurement results of transgenic lines for heterologous expression MfWRKY47 Measurement results of O 2- content and NBT staining results of transgenic lines for heterologous expression

[0037] Figure 15 Measurement results of antioxidant enzyme (SOD), peroxidase (POD), catalase (CAT) activities and malondialdehyde (MDA) content of plants with heterologous expression under drought stress MfWRKY47 ;

[0038] Figure 16 Measurement results of antioxidant enzyme (SOD), peroxidase (POD), catalase (CAT) activities and malondialdehyde (MDA) content of plants with heterologous expression under salt stress MfWRKY47 ;

[0039] Figure 17 Results of regulating related gene expression of plants with heterologous expression under drought stress MfWRKY47 ;

[0040] Figure 18 Results of regulating related gene expression of plants with heterologous expression under salt stress MfWRKY47 ; Detailed implementation methods

[0041] The present invention provides a Medicago falcata WRKY47 transcription factor, the amino acid sequence of which is as shown in SEQ ID NO: 1 (MLNLVNDRCNVLQNRLLLAMHMHQSSSLPQNNHNLLLKGNTQDAEKPVLPTRQFFDEPSPSDCSKNNGFAIVENNENNMGRNLACEYINEGEINSKIEDQSSEVGCRRARVSIRARSDFAFMVDGCQWRKYGQKTAKGNPCPRAYYRCSMGTSCPVRKQVQRCFKDESVFITTYEGNHNHQLPPAAKPIANLTSSALNTFLPTSSTNLQQYGNNLTNTFLFSSPLSPPNSNAIATFSPSPTCPTITLDFTLPPSNYLQFKNHKQSSLLPFPFQGHYPPSFEVFPNLINNERKLDLVDVVSEALEKDPSLKEALFSAMSSFTNGDSSNINNQSQLPSKSSG).

[0042] In the present invention, the Medicago falcata WRKY47 transcription factor improves the salt tolerance and / or drought tolerance of plants through positive regulation. The subcellular localization results show that the Medicago falcata WRKY47 transcription factor is localized in the nucleus.

[0043] The present invention provides a gene encoding the Medicago falcata WRKY47 transcription factor.

[0044] In the present invention, the nucleotide sequence of the gene is preferably as shown in SEQ ID NO: 2. It should be understood that considering the degeneracy of codons, modifying the nucleotide sequence of the gene without changing the amino acid sequence also falls within the protection scope of the present invention.

[0045] The expression level of the gene of the present invention is positively correlated with the salt tolerance and / or drought tolerance of plants. The examples of the present invention show that MfWRKY47 it is expressed in the roots, stems and leaves of Medicago falcata, but the expression is the highest in the roots. At the same time, MfWRKY47 the expression level can respond to the changes in the salt concentration and drought environment in the growth environment, and the expression level MfWRKY47 increases in the salt environment or drought environment. The present invention further constructs heterologous expression MfWRKY47 Arabidopsis thaliana plants and detects their salt tolerance and drought tolerance. The results show that the Medicago falcata MfWRKY47 gene effectively improves the salt tolerance and drought tolerance of Arabidopsis thaliana, specifically manifested as under salt stress or drought stress conditions, the expression MfWRKY47The seeds of Arabidopsis thaliana plants have an increased germination rate compared to wild-type plants, with improvements in aspects such as root length and fresh weight, and also an increase in chlorophyll content, while the accumulation of H2O2 and O 2- is reduced, and the antioxidant enzyme activity is increased. In addition, the malondialdehyde content is decreased, indicating that the expression of MfWRKY47 in Arabidopsis thaliana plants enhances stress resistance. At the same time, differentially expressed genes in heterologously expressed MfWRKY47 Arabidopsis thaliana plants were analyzed. The results showed that under drought stress, heterologous expression of MfWRKY47 significantly upregulated stress-responsive genes LEA4-5 , peroxidase genes PRX52 , and cation transporter genes CAX3 . Under salt stress, heterologous expression of MfWRKY47 significantly upregulated nitrogen metabolism regulatory genes NIA2 , Na + / H + transporter genes NHX4 , and salt stress-responsive genes CHX16 . This indicates that the MfWRKY47 gene regulates the expression of stress-related genes, antioxidant enzyme genes, and ion transporter genes to enhance the salt and drought tolerance of plants.

[0046] The present invention provides a gene-derived product containing the said gene, including an expression cassette containing the said gene; the expression cassette further contains a promoter, and the nucleotide sequence of the promoter is as shown in SEQ ID NO: 3.

[0047] In the present invention, the promoter contains cis-acting elements responsive to drought stress and osmotic stress, and can respond to changes in salt concentration and / or drought environment. Examples of the present invention show that under salt stress and / or drought stress conditions, the promoter drives GUS expression, indicating that the promoter is induced by salt stress and / or drought stress. The promoter can be applied to drive the expression of plant genes in plants, to prepare salt-tolerant and / or drought-tolerant transgenic plants, and for one or more of plant genetic breeding or germplasm improvement.

[0048] The present invention provides an application of the Medicago falcata WRKY47 transcription factor, the said gene, or the said gene-derived product in at least one of the following works: 1) improving the salt tolerance and / or drought tolerance of plants; 2) creating salt-tolerant and / or drought-tolerant plant varieties.

[0049] In the present invention, the Medicago falcata WRKY47 transcription factor or the gene preferably enhances the salt tolerance and / or drought tolerance of plants through positive regulation. The salt tolerance preferably includes the resistance of plants to salt environments. The salt in the salt environment preferably includes sodium chloride. The concentration of sodium chloride is preferably above 50 mM, more preferably 100 - 200 mM, and most preferably 150 mM. The drought tolerance preferably includes the resistance of plants to drought environments. The drought environment is preferably a drought environment simulated by mannitol, and the concentration of mannitol is preferably above 100 mM, more preferably 150 - 300 mM, and most preferably 200 mM.

[0050] In the present invention, the enhancement of the salt tolerance and / or drought tolerance of plants preferably includes changes in at least one of the following indicators: increasing plant biomass, increasing the chlorophyll content of plants, reducing the accumulation of H2O2 and / or O 2- accumulation in plants, increasing the antioxidant enzyme activity of plants, and reducing the malondialdehyde content of plants.

[0051] In the present invention, the plants preferably include Medicago plants or Arabidopsis plants. More preferably, they include at least one of the following: Medicago falcata, Medicago sativa, and Arabidopsis thaliana. The Medicago plants include Medicago falcata, Medicago lupulina, Medicago arabica, and Medicago hispida. The Arabidopsis plants preferably include Arabidopsis thaliana. Since Arabidopsis thaliana is a model organism, in the examples of the present invention, it was verified in Arabidopsis thaliana that the Medicago falcata WRKY47 transcription factor or the gene has the effect of enhancing the salt tolerance and / or drought tolerance of plants, and it can be obtained that within the range of dicotyledonous plants, it has the biological effect of enhancing the salt tolerance and / or drought tolerance of plants.

[0052] The present invention provides a method for enhancing the salt tolerance and / or drought tolerance of plants by overexpressing the Medicago falcata WRKY47 transcription factor or the gene-derived product in plants.

[0053] In the present invention, the gene-derived products include recombinant vectors and / or recombinant bacteria. The backbone vector of the recombinant vector preferably includes pPZP221 (35s-nos), and the host bacteria of the recombinant bacteria preferably include Agrobacterium, more preferably Agrobacterium tumefaciens GV3101. The present invention does not limit the method for transferring into plants, and conventional methods for transforming plants in the art can be used. The plants are preferably the same as those in the above application and will not be elaborated here. In the examples of the present invention, MfWRKY47 By genetic transformation of Arabidopsis thaliana through the floral dip method, the obtained heterologous expression MfWRKY47 Arabidopsis thaliana has significantly improved salt tolerance and / or drought tolerance compared to wild-type Arabidopsis thaliana.

[0054] The invention provides a method for identifying plant salt tolerance and / or drought resistance, which comprises detecting the expression level of the alfalfa WRKY47 transcription factor or the gene in the plant, and judging the plant salt tolerance and / or drought resistance according to the expression level: the expression level of the alfalfa WRKY47 transcription factor or the gene is positively correlated with the plant salt tolerance and / or drought resistance.

[0055] In the present invention, the reagent for detecting the expression level of the gene preferably includes a forward primer as shown in SEQ ID NO:4 and a reverse primer as shown in SEQ ID NO:5.

[0056] The embodiment of the present invention will MfWRKY47 Heterologous expression obtained by transformation of Arabidopsis MfWRKY47 Arabidopsis thaliana, detected by the reagents indicated heterologous expression MfWRKY47 Arabidopsis MfWRKY47 High expression ， And the salt tolerance and / or drought resistance of the plant are significantly improved compared with the wild-type Arabidopsis. Therefore, the salt tolerance and / or drought resistance of the plant can be judged according to the expression level of the WRKY47 transcription factor of Medicago truncatula or the gene in the plant.

[0057] In order to further illustrate the present invention, the scheme provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0058] The information of the primers used in the examples of the present invention is shown in Table 1, and the bold parts in the sequences represent restriction enzyme cleavage sites.

[0059] Table 1 Primer names and nucleotide sequence information

[0060]

[0061] Example 1

[0062] Acquisition of MfWRKY47 transcription factor from Medicago truncatula

[0063] 1. Obtaining hydroponic seedlings of yellow alfalfa

[0064] Wild yellow alfalfa seeds were collected from Xilin Gol grassland. Full seeds were selected and vernalized at 4°C for 2 weeks. The seeds were gently sanded with sandpaper to break the skin, soaked in 75% alcohol for 8 minutes, sterilized with 0.1% mercuric chloride for 5 minutes, rinsed with sterile water 5 times, and soaked for 8 hours. They were sown on 1 / 2 MS medium and placed in a light incubator (25±1°C, 40 μmol·m -2 ·s -1, cultured under 16 h light / 8 h darkness), after 1 week, select seedlings with consistent growth and transplant them into a culture box filled with 1 / 2 Hoagland nutrient solution, and culture them in a greenhouse (25±1°C, 16 h light / 8 h darkness) to obtain hydroponic seedlings as Figure 1 shown.

[0065] 2. Obtaining of Medicago falcata MfWRKY47 transcription factor

[0066] Using 4-week-old hydroponic seedlings of Medicago falcata as materials, collect leaves, after grinding with liquid nitrogen, use Trizol reagent (purchased from TaKaRa) to extract total RNA. Then use a reverse transcription kit (purchased from TaKaRa, catalog number 6210A) to reverse transcribe and synthesize cDNA. Using cDNA as a template, use primers MfWRKY47 -R and MfWRKY47 -F for RT-PCR amplification, and the amplification conditions are 98 °C for 5 min; 95 °C for 1 min, 56 °C for 30 s, 68 °C for 1 min, 30 cycles; 68 °C for 10 min. After the PCR reaction is completed, use a PCR product purification kit (purchased from Sangon Biotech Co., Ltd.) to purify the target fragment and then ligate it with pMD19-T to construct pMD -MfWRKY47 , after ligation reaction at 16 °C for 3 h, transform it into competent cells Trans1-T1, and after identification by colony PCR (the primers for colony PCR are MfWRKY47 -R and MfWRKY47 -F) and enzyme digestion verification, the CDS sequence of the MfWRKY47 transcription factor gene is obtained through sequencing analysis. MfWRKY47 The nucleotide sequence of the CDS of the gene is shown in SEQ ID NO: 2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 1.

[0067] Use MEGA software to construct an evolutionary tree by the maximum likelihood method, and set the Bootstrap value to 1000. The verification analysis of the evolutionary tree is as Figure 2 shown: MfWRKY47 has the highest homology with AtWRKY47.

[0068] Example 2

[0069] Medicago falcata MfWRKY47 Obtaining of the MfWRKY47p sequence of the gene promoter

[0070] Using 4-week-old hydroponic seedlings of Medicago falcata in Example 1 as materials, collect leaves, after grinding with liquid nitrogen, use the CTAB method to extract the genomic DNA of Medicago falcata, and then use the GenomeWalker kit (TaKaRa) to construct a GenomeWalker DNA library. According to the obtainedMfWRKY47 The nucleotide sequence of the gene CDS is designed as follows WRKY47 p-GSP1 and WRKY47 p-GSP2 primers. Using the GenomeWalker DNA library as a template, two rounds of PCR amplification were carried out. The conditions for the first round of PCR were: 94 °C for 25 s, 72 °C for 3 min, 7 cycles; 94 °C for 25 s, 67 °C for 3 min, 32 cycles; 67 °C for 10 min; 12 °C for 10 min. The conditions for the second round of PCR were: 94 °C for 25 s, 72 °C for 3 min, 5 cycles; 94 °C for 25 s, 67 °C for 3 min, 20 cycles; 67 °C for 7 min; 12 °C for 10 min. After purification, the PCR products were ligated with pMD19-T and introduced into Trans1-T1, and DNA sequences were obtained by sequencing analysis. Based on the obtained DNA sequences, WRKY47 p-GSP3 and WRKY47 p-GSP4 primers were designed. Using the GenomeWalker DNA library as a template, the second-step PCR walking amplification was carried out. After purification, the PCR products were ligated with pMD19-T and introduced into Trans1-T1, and DNA sequences were obtained by sequencing analysis. The DNA sequences obtained by the secondary walking amplification were spliced to obtain the promoter sequence DNA information. Based on the obtained promoter sequence DNA information, specific primers WRKY47p-R and WRKY47p-F were designed, and PCR amplification was carried out using the Medicago falcata genome DNA as a template. The PCR amplification conditions were 98 °C for 5 min; 98 °C for 1 min, 57 °C for 30 s, 68 °C for 1 min, 30 cycles; 68 °C for 10 min. After purification, the PCR products were ligated with pMD19-T to form the recombinant plasmid pMD-WRKY47p and introduced into Trans1-T1, and the WRKY47p DNA sequence was obtained by sequencing analysis.

[0071] Medicago falcata MfWRKY47 The nucleotide sequence of the promoter MfWRKY47p of the Medicago falcata gene is shown in SEQ ID NO:3. Using PlantCARE (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) for prediction and analysis, the results showed that it contains cis-acting elements such as response to drought stress and osmotic stress.

[0072] Example 3

[0073] Medicago falcata MfWRKY47 Analysis of gene expression pattern

[0074] 1. Analysis of tissue expression characteristics pattern

[0075] Using 4-week-old Medicago falcata hydroponic seedlings in Example 1 as materials, root, stem, and leaf tissues were collected respectively. After grinding in liquid nitrogen, total RNA was extracted using Trizol reagent (TaKaRa). Then, cDNA was reverse-transcribed using a reverse transcription kit (PrimeScript™ RT reagent Kit with gDNA Eraser Kit, TaKaRa). Using WRKY47 -qR and WRKY47 -qF as primers, Real-time PCR analysis was performed using a fluorescence quantitative kit (TB Green™ Premix Ex Taq™ II, TaKaRa). The experiment used MfEF1α- qR and MfEF1α- qF primers to detect MfEFlα as the internal reference gene.

[0076] The results were as Figure 3 shown: MfWRKY47 It was expressed in the roots, stems, and leaves of Medicago falcata, but the expression was highest in the roots, and the expression level in the roots was significantly higher than that in the stems and leaves.

[0077] 2. Analysis of expression patterns in response to salt and drought stresses

[0078] Take 4-week-old Medicago falcata hydroponic seedlings in Example 1 and divide them into a salt stress treatment group and a drought stress treatment group. The salt stress treatment group was to culture 4-week-old Medicago falcata hydroponic seedlings in Hoaglands nutrient solution containing 200 mM NaCl. The drought stress treatment group was to culture 4-week-old Medicago falcata hydroponic seedlings in Hoaglands nutrient solution containing 200 mM mannitol.

[0079] At 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h of treatment, roots, stems, and leaves were collected respectively, RNA was extracted, and cDNA was reverse-transcribed using a reverse transcription kit (PrimeScript™ RT reagent Kit with gDNA Eraser Kit, TaKaRa). Using WRKY47-qR and WRKY47-qF as primers, Real-time PCR analysis was performed using a fluorescence quantitative kit (TB Green™ Premix Ex Taq™ II, TaKaRa). The experiment used MfEFlα- qR and MfEFlα- qF primers to detect MfEFlα as the internal reference gene.

[0080] The real-time PCR detection system is as follows: 10.0 μL of TB Green Premix Ex Taq II (2×), 0.8 μL of WRKY47-qR (10 μM), 0.8 μL of WRKY47-qF (10 μM), 2.0 μL of cDNA, and 6.4 μL of RNase-free Water.

[0081] The reaction conditions are as follows: 95 °C for 1 min; 95 °C for 5 s, 58 °C for 30 s, 72 °C for 20 s, for 40 cycles; melting curve from 65 °C to 95 °C, lasting 5 s at every 0.5 °C.

[0082] The results of the drought stress treatment group are as Figure 4 shown in A: After 1 h of drought stress treatment, MfWRKY47 the expression level in roots increased, the expression levels in stems and leaves increased significantly, and at 12 h MfWRKY47 the expression level in roots reached the highest value, and at 24 h MfWRKY47 the expression level in stems reached the highest value, and at 6 h MfWRKY47 the expression level in leaves reached the highest value. It can be seen that MfWRKY47 the expression in roots, stems and leaves of Medicago falcata is induced by drought stress.

[0083] The results of the salt stress treatment group are as Figure 4 shown in B: After 1 h of salt stress treatment, MfWRKY47 the expression levels in roots, stems and leaves increased significantly, and at 3 h MfWRKY47 the expression level in roots reached the highest value. It can be seen that MfWRKY47 the expression in roots, stems and leaves of Medicago falcata is induced by salt stress.

[0084] 3. Medicago falcata MfWRKY47 Analysis of the GUS expression pattern driven by the WRKY47p promoter of the gene

[0085] Construction of the expression vector and infiltration of Arabidopsis thaliana:

[0086] The vector pORE R1 STOCK: CD3-929 was purchased from the Arabidopsis Biological Resource Center (ABRC). The vector pORE R1 and the recombinant plasmid pMD-WRKY47p in Example 2 were respectively Bam digested with H Ⅰ and SacI digestion, the target fragment was recovered, and the expression vector pORER1::WRKY47p-GUS was constructed with the vector pORER1 as the backbone. The expression vector pORER1::WRKY47p-GUS was introduced into Agrobacterium GV3101 by freeze-thaw method. Arabidopsis thaliana was genetically transformed by inflorescence dip method, and the transformed Arabidopsis thaliana T2 generation seeds were obtained by 40 mg / mL Kan screening and culture.

[0087] GUS staining and GUS activity analysis:

[0088] The above T2 generation seeds were sterilized and sown on 1 / 2 MS solid medium. After culturing for 12 days, the seedlings with the same growth were selected and divided into salt stress treatment group, drought stress treatment group and control group. The salt stress treatment group was transferred to 1 / 2 MS medium containing 150 mM NaCl for 2 days; the drought stress treatment group was transferred to 1 / 2 MS medium containing 100 mM Minnitol for 2 days; the seedlings grown in 1 / 2MS solid medium were treated as control group (CK) for 2 days. GUS staining was performed after 2 days, and the coloring distribution of GUS in each group was observed under a stereo microscope (NIKON SMZ18). At the same time, the β-glucuronidase detection kit was used to detect GUS activity.

[0089] The results are as follows Figure 5 As shown: blue was observed in both the salt stress treatment group and the drought stress treatment group, and the GUS activity was significantly increased as determined by the β-glucuronidase detection kit. MfWRKY47 The promoter-driven GUS expression is induced by salt stress and drought stress.

[0090] Example 4

[0091] Yellow clover MfWRKY47 Subcellular localization analysis of gene-encoded proteins

[0092] 1. Vector construction and identification methods

[0093] Design contains Sal I and Kpn The primers pCAM-MfWRKY47-F and pCAM-MfWRKY47-R at the I site were used to construct the recombinant plasmid pMD -MfWRKY47 As a template, PCR amplification of MfWRKY47 The amplification reaction conditions were as follows: 98 °C for 5 min; 95 °C for 1 min, 55 °C for 30 s, 72 °C for 1 min, 30 cycles; 68 °C for 10 min. The PCR products were purified to obtain the purified products. Sal I andKpn Ⅰ After digesting the above purified product and the pCAMBIA1300-35S-EGFP vector (purchased from Wuhan Miaoling Biotechnology Co., Ltd.), the linearized vector and the target fragment were purified and then ligated with T4 DNA Ligase at 16 °C for 12 h to construct the pCAMBIA1300(35S:: MfWRKY47 -EGFP) vector. After being introduced into Trans1-T1 and analyzed by sequencing, the positive clones verified by sequencing were cultured on a large scale, and the pCAMBIA1300(35S:: MfWRKY47 -EGFP) vector was extracted. The Agrobacterium tumefaciens GV3101 was transformed by the freeze-thaw method to obtain the GV3101 bacterial liquid transformed with the pCAMBIA1300(35S:: MfWRKY47 -EGFP) vector.

[0094] 2. Transient expression in Nicotiana benthamiana leaves

[0095] Take 200 μL of the GV3101 bacterial liquid transformed with the pCAMBIA1300(35S:: MfWRKY47 -EGFP) vector into 10 mL of YEB medium and culture it overnight at 28 °C. Pipette 100 μL of the overnight cultured bacterial liquid into 30 mL of YEB medium, and at the same time add 20 μL of AS (200 μM) and 100 μL of MES (10 mM), and culture at 28 °C until the OD 600 of the bacterial liquid = 1.2 - 1.6. Centrifuge at 4 °C and 4400 rpm for 10 min to precipitate the bacteria. The bacteria were resuspended with an appropriate amount of osmotic solution (10 mM MgCl2, 10 mM MES and 200 μM AS) to an OD 600 = 0.8 - 1.0, and cultured at room temperature in the dark for 3 h. The cultured bacterial liquid was injected into the lower epidermis of tobacco leaves and cultured under weak light for 48 h.

[0096] 3. Observation by laser confocal microscope

[0097] Under light avoidance conditions, cut the tobacco leaves and place them in the DAPI (5 μg / mL) staining solution. After 15 min, rinse with 1×PBS buffer solution. After rinsing 2 times, place the leaves face up on the glass slide for making slides, and observe with a laser confocal microscope. The experiment used the injection of the GV3101 bacterial liquid containing the pCAMBIA1300-35S-EGFP vector as a control.

[0098] The results are as Figure 6 shown: The protein encoded by the Medicago falcata MfWRKY47 gene is localized in the nucleus.

[0099] Example 5

[0100] Medicago falcataMfWRKY47 Verification of the Transcription Activation Function of the Gene-Encoded Protein

[0101] The vector pGBKT7 (purchased from Takara) and the recombinant plasmid pMD in Example 1 -MfWRKY47 were respectively digested with Eco R Ⅰ and Sal I, and the target fragments were recovered. Then they were ligated with T4 DNA Ligase at 16 °C for 12 h to construct the vector pGBKT7 - MfWRKY47 After that, the yeast strain AH109 was transformed by the LiAc method (Yeast Transformation Kit) and spread on SD / Trp - solid medium, and single colonies were obtained by culturing at 30 °C. After picking single colonies and identifying them by colony PCR, they were cultured in SD / Trp - medium at 30 °C until the OD6 600 of the bacterial liquid = 0.5, and then diluted 10 -1 times, 10 -2 times, and 10 -3 times in sequence. 15 μL of each diluted bacterial liquid was respectively dropped on SD / Trp- and SD / Trp- / His- / X-α-Gal (5 mg / mL) media, and cultured at 30 °C for 3 d to observe the colony growth status and α-Gal activity. The experiment used the transformed pGBKT7-53 + pGADT7-T bacterial cells as the positive control, and the transformed pGBKT7-Lam + pGADT7-T bacterial cells and the transformed pGBKT7 bacterial cells as the negative controls.

[0102] The results are as Figure 7 shown: The gene-encoded protein of Medicago falcata MfWRKY47 was not detected with transcription activation function in yeast cells.

[0103] Example 6

[0104] Verification of the Binding of the Gene-Encoded Protein of Medicago falcata MfWRKY47 to the W-box

[0105] 1. Construction of the Bait Vector

[0106] Three oligonucleotide sequences (SEQ ID NO:25) containing three W-boxes with Sac I and Sal I restriction sites and three oligonucleotide sequences (SEQ ID NO:26) of three mW-boxes with mutation sites were designed and synthesized. After annealing them respectively to synthesize double-stranded DNA, they were respectively ligated with the vector that had been digested with Sac I and SalThe pAbAi vector digested with I (purchased from Takara) was ligated, transformed into Trans1-T1, and after sequencing analysis, the bait vectors pAbAi-W-box and pAbAi-mW-box were obtained.

[0107] 2. Construction of expression vector

[0108] Primers pAD-MfWRKY47-F and pAD-MfWRKY47-R containing EcoR Ⅰ, Sac Ⅰ restriction sites were designed. Using the recombinant plasmid pMD in Example 1 -MfWRKY47 as a template, the target fragment was amplified by PCR. The amplification reaction conditions were: 98 °C for 5 min; 95 °C for 1 min, 57 °C for 30 s, 72 °C for 1 min, for 30 cycles; 68 °C for 10 min. After digestion with EcoR Ⅰ, Sac Ⅰ and purification of the PCR product and the pGADT7 vector (purchased from Takara), T4 DNA Ligase was used to ligate at 16 °C for 12 h to construct the pGADT7-MfWRKY47 vector, which was introduced into Trans1-T1. After verification by colony PCR and restriction digestion, the positive plasmid vector was obtained.

[0109] 3. Verification of binding characteristics

[0110] pAbAi-W-box and pAbAi-mW-box were linearized with BstB I respectively. After purification, 1 μg was taken and transformed into the yeast strain Y1HGold by the LiAc method. After identification by colony PCR using the primers W-box, pAbAi-R and mW-box, pAbAi-R in Table 1 respectively, positive strains were obtained, denoted as W-Y1HGold and mW-Y1HGold respectively. The cells of W-Y1HGold and mW-Y1HGold were respectively spread on SD / Ura- solid medium containing 0 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 350 ng / mL, 400 ng / mL, 450 ng / mL and 500 ng / mL AbA for culture, and the lowest AbA concentration that completely inhibited the growth of the bait strain was determined to be 350 ng / mL.

[0111] The pGADT7 and pGADT7-MfWRKY47 plasmids were respectively transformed into the cells of W-Y1HGold and mW-Y1HGold by the LiAc method, inoculated into SD / Ura-Leu- medium, cultured overnight at 30 °C, and then the cell suspension was adjusted to OD with sterile 0.9% NaCl solution 600= 0.5, and then serially diluted 10 -1 、10 -2 and 10 -3 times. Take 15 µL of each diluted bacterial solution and drop it on the SD / Ura-Leu- / AbA (350 ng / mL) medium, and culture at 30 °C for 3 d to observe the colony growth status.

[0112] The results are as Figure 8 shown: The protein encoded by the Medicago falcata MfWRKY47 gene can bind to the W-box.

[0113] Example 7

[0114] Medicago falcata MfWRKY47 Analysis of the application of the gene in regulating plant salt tolerance and drought resistance

[0115] 1. Heterologous expression MfWRKY47 Method for creating transgenic Arabidopsis thaliana lines

[0116] Using Arabidopsis thaliana plants as test plants, design primers pPZP221- Bam HⅠ, Sac Ⅰ restriction enzyme sites, and use the recombinant plasmid pMD MfWRKY47 -F and pPZP221- MfWRKY47 -R in Example 1 as a template for PCR amplification of the target fragment. The amplification reaction conditions are: 98 °C for 5 min; 95 °C for 1 min, 57 °C for 30 s, 72 °C for 1 min, 30 cycles; 68 °C for 10 min. After the amplification is completed, the PCR product is obtained. After -MfWRKY47 digesting and purifying the PCR product and the pPZP221 (35s-nos) vector with Bam HⅠ, Sac Ⅰ, T4 DNA Ligase is used to ligate at 16 °C for 12 h to construct a heterologous expression vector pPZP221 (35s- MfWRKY47 - nos), which is introduced into Trans1-T1. After verification by colony PCR and enzyme digestion, a positive plasmid vector is obtained. The heterologous expression vector pPZP221 (35s- MfWRKY47 - nos) is introduced into Agrobacterium tumefaciens GV3101 by the freeze-thaw method. Arabidopsis thaliana is genetically transformed by the floral dip method, and the transgenic T3 generation homozygous lines of Arabidopsis thaliana are obtained after screening and culturing with 50 mg / L Gen. The pPZP221 (35s-nos) vector is the same as the pPZP221 (35s-nos) vector in the prior art (Niu Yiding. Research on the DREB-like transcription factor gene of Medicago sativa [D]. Inner Mongolia University, 2008.).

[0117] Fresh leaves of the homozygous lines of transgenic Arabidopsis thaliana T3 generation were taken to extract RNA, and the obtained cDNA after reverse transcription was used as a template. Using WRKY47 -qR and WRKY47 -qF primers, real-time PCR analysis was performed using a fluorescence quantitative kit (TB Green™ Premix Ex Taq™ II, TaKaRa). The experiment used Atactin2 as the internal reference gene.

[0118] The real-time PCR detection system was: TB Green Premix Ex Taq II (2×) 10.0 μL, WRKY47-qR (10 μM) 0.8 μL, WRKY47-qF (10 μM) 0.8 μL, cDNA 2.0 μL, RNase-free Water 6.4 μL.

[0119] The reaction conditions were: 95 °C for 1 min; 95 °C for 5 s, 58 °C for 30 s, 72 °C for 20 s, 40 cycles; melting curve from 65 °C to 95 °C, lasting 5 s every 0.5 °C.

[0120] The results were as Figure 9 shown: The MfWRKY47 gene was highly expressed in all transgenic lines. The MfWRKY47 gene expression levels in the OE-8 transgenic line, OE-9 transgenic line, and OE-12 transgenic line were higher than those in the OE-3 transgenic line and OE-15 transgenic line. The OE-8 transgenic line, OE-9 transgenic line, and OE-12 transgenic line with higher expression levels were selected for subsequent experiments.

[0121] 2. Verification experiments on salt tolerance and drought resistance of transgenic plants

[0122] 1) Analysis of seed germination rate

[0123] The seeds were divided into a salt stress treatment group and a drought stress treatment group. The drought stress treatment group sowed the T3 generation seeds of the wild type and transgenic lines in 1 / 2 MS medium containing Mannitol (100 mM, 200 mM, and 300 mM) respectively; the salt stress treatment group sowed the T3 generation seeds of the wild type and transgenic lines in 1 / 2 MS medium containing NaCl (50 mM, 100 mM, and 150 mM) respectively. 30 seeds of each line were sown and cultured in an artificial climate incubator. After 7 d, the seed germination rate was counted and photographed. When the radicle broke through the seed coat by 2 mm, it was regarded as germination. The experiment was designed with 3 biological replicates and 3 technical replicates.

[0124] 2) Analysis of seedling growth indexes

[0125] The T3-generation seeds of wild-type and transgenic lines were sown in 1 / 2 MS medium. After 4 days of cultivation, the seedlings with consistent growth vigor were selected and divided into a salt stress treatment group and a drought stress treatment group. The drought stress treatment group was transferred to 1 / 2 MS medium containing Mannitol (100 mM, 200 mM, and 300 mM), and the salt stress treatment group was transferred to 1 / 2 MS medium containing NaCl (50 mM, 100 mM, and 150 mM). After vertical cultivation for 10 days, the root lengths and fresh weights of different lines were measured using a scale with a precision of 0.1 cm and a precision electronic balance of 0.1 mg, and photos were taken. All experiments were designed with 3 biological replicates and 3 technical replicates.

[0126] Figure 10 The results showed that under salt stress conditions, when the NaCl concentration was 100 mM and 150 mM, the germination rate of the transgenic lines was significantly higher than that of the wild-type. When the NaCl concentration was 50 mM, 100 mM, and 150 mM, the primary root length and plant fresh weight of the transgenic lines were significantly higher than those of the wild-type. Under drought stress conditions, when the Mannitol concentration was 100 mM, 200 mM, and 300 mM, the germination rate, primary root length, and plant fresh weight of the transgenic lines were significantly higher than those of the wild-type.

[0127] 3) Analysis of physiological and biochemical indexes of salt tolerance and drought resistance

[0128] The T3-generation seeds of wild-type and transgenic lines were sown in 1 / 2 MS medium. After 14 days of cultivation, they were transplanted into nutrient soil and conventionally cultivated for 3 weeks, and then divided into a salt stress treatment group and a drought stress treatment group. The drought stress treatment group was subjected to drought stress by stopping watering for 10 days; the salt stress treatment group was subjected to salt stress by irrigating Arabidopsis thaliana seedlings with distilled water containing 50 mM, 100 mM, and 150 mM NaCl at 3-day intervals; Arabidopsis thaliana irrigated with distilled water without stress treatment was used as a control (the control was irrigated with distilled water). After 10 days, the leaves of Arabidopsis thaliana of each line after the above treatments were collected to measure the chlorophyll content, H2O2 and O 2- concentration, detect the activities of its antioxidant enzymes (SOD), peroxidase (POD), and catalase (CAT), and the content of malondialdehyde (MDA), and measure the leaf water loss rate of the drought stress treatment group. All experiments were designed with 3 biological replicates and 3 technical replicates.

[0129] Figure 11 The results showed that the leaf water loss rate of the transgenic lines after drought stress treatment was significantly lower than that of the wild-type. Figure 12 The results showed that the chlorophyll content of the transgenic lines after salt stress treatment or drought stress treatment was significantly higher than that of the wild-type. Figure 13The results showed that the H2O2 accumulation in transgenic lines was significantly reduced compared with that in the wild type after salt stress treatment or drought stress treatment. Figure 14 The results showed that the O 2- accumulation in transgenic lines was significantly reduced compared with that in the wild type after salt stress treatment or drought stress treatment. Figure 15 The results showed that after drought stress treatment, the activities of SOD, POD and CAT in transgenic lines were significantly increased compared with those in wild-type plants, and the content of malondialdehyde (MDA) was significantly reduced. Figure 16 The results showed that after salt stress treatment, the activities of SOD, POD and CAT in transgenic lines were significantly increased compared with those in wild-type plants, and the content of malondialdehyde (MDA) was significantly reduced.

[0130] The above results showed that Medicago falcata MfWRKY47 genes could be applied to improve the germination rate of plant seeds under salt and drought stress conditions, promote the growth of seedlings ( Figure 10 ), and participate in regulating the salt tolerance and drought resistance of plants by reducing the leaf water loss rate ( Figure 11 ), reducing chlorophyll degradation ( Figure 12 ), reducing the accumulation of H2O2 and O 2- in plant leaves ( Figure 13 and Figure 14 ), increasing the antioxidant enzyme activity and reducing the degree of membrane damage ( Figure 15 and Figure 16 ).

[0131] 3. Medicago falcata MfWRKY47 Gene regulation analysis of salt tolerance and drought resistance related genes

[0132] RNA-seq analysis of transgenic lines (OE-12 transgenic lines, denoted as OE in Figure 17 and Figure 18 ), after differential expression gene analysis by DEG, using AtActin1 as the internal reference gene, RT-qPCR verification was carried out, and the results are shown in Figure 17 and Figure 18 . Figure 17 The results showed that the expression levels of stress response genes LEA4-5 , peroxidase genes PRX52 , cation transporter genes CAX3 in transgenic lines were significantly increased compared with those in the wild type. Therefore, heterologous expression of MfWRKY47 under drought stress could significantly up-regulate the expression of stress response genes LEA4-5 , peroxidase genes PRX52 , cation transporter genes CAX3 . Figure 18 The results showed that the expression levels of nitrogen metabolism regulatory genes NIA2 , Na+ / H + Transporter gene NHX4 , salt stress-responsive gene CHX16 showed significantly increased expression levels compared to the wild type. Therefore, heterologous expression under salt stress MfWRKY47 could significantly up-regulate the nitrogen metabolism regulatory gene NIA2 , Na + / H + transporter gene NHX4 , salt stress-responsive gene CHX16 expression. The above results indicate that MfWRKY47 genes can be applied to regulate the expression of stress-related genes, antioxidant enzyme genes, and ion transporter genes to enhance the salt tolerance and drought resistance of plants.

[0133] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments can be obtained based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Medicago falcata WRKY47 transcription factor, characterized in that, The amino acid sequence is as shown in SEQ ID NO:

1.

2. A gene encoding the Medicago falcata WRKY47 transcription factor according to claim 1.

3. A gene-derived product comprising the gene according to claim 2, characterized in that, Comprising an expression cassette containing the gene according to claim 2; The expression cassette further comprises a promoter, and the nucleotide sequence of the promoter is as shown in SEQ ID NO:

3.

4. Use of the Medicago falcata WRKY47 transcription factor according to claim 1, the gene according to claim 2 or a gene-derived product according to claim 3 in at least one of the following works: 1) Improving the salt tolerance and / or drought resistance of plants; 2) Creating salt-tolerant and / or drought-resistant plant varieties; The method for improving the salt tolerance and / or drought resistance of plants is to overexpress the gene in plants; The method for creating salt-tolerant and / or drought-resistant plant varieties is to overexpress the gene in plants; The plant is Arabidopsis thaliana.

5. The application according to claim 4, wherein The Medicago falcata WRKY47 transcription factor or the gene improves the salt tolerance and / or drought resistance of plants through positive regulation.

6. The application according to claim 4, characterized in that The improvement of the salt tolerance and / or drought tolerance of plants includes changes in at least one of the following indicators: an increase in plant biomass, an increase in the chlorophyll content of plants, a decrease in the accumulation of H2O2 and / or O 2- accumulation in plants, an increase in the antioxidant enzyme activity of plants, and a decrease in the malondialdehyde content of plants.

7. A method for improving the salt tolerance and / or drought tolerance of plants, characterized in that, Overexpressing the Medicago falcata WRKY47 transcription factor according to claim 1, the gene according to claim 2 or a gene-derived product according to claim 3 in plants; The plant is Arabidopsis thaliana.

8. A method for identifying the salt tolerance and / or drought tolerance of plants, characterized in that, Detecting the expression level of the Medicago falcata WRKY47 transcription factor according to claim 1 or the gene according to claim 2 in plants, and judging the salt tolerance and / or drought resistance of plants according to the expression level: The expression level of the Medicago falcata WRKY47 transcription factor or the gene is positively correlated with the salt tolerance and / or drought resistance of plants; The plant is Arabidopsis thaliana.

9. The method according to claim 8, wherein The reagents for detecting the expression level of the gene include the forward primer shown in SEQ ID NO: 4 and the reverse primer shown in SEQ ID NO: 5.

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