Medicago falcata WRKY47 transcription factor, gene and application of transcription factor in improvement of salt tolerance and / or drought resistance of plants

Abstract

The invention provides a medicago falcata WRKY47 transcription factor, a gene and application of the transcription factor to improvement of salt tolerance and / or drought resistance of plants, and belongs to the technical field of biological agriculture. The invention provides a medicago falcata WRKY47 transcription factor. The amino acid sequence of the medicago falcata WRKY47 transcription factor is shown as SEQ ID NO: 1. The medicago falcata WRKY47 transcription factor provided by the invention can improve the salt tolerance and / or drought resistance of plants through forward regulation and control. The embodiment of the invention shows that the expression quantity of the MfWRKY47 can respond to the change of salt concentration and drought degree in a growth environment, and the salt tolerance and drought resistance of a transgenic plant are obviously improved compared with those of a wild plant by heterologous expression of the MfWRKY47. The medicago falcata WRKY47 transcription factor enriches a salt resistance and / or drought resistance genetic resource library, and has important practical application significance in stress-resistant plant strain breeding.

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 applications 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, receptors, etc. to adapt to corresponding adversity stresses. 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, 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 resistance-related functional genes of Medicago falcata, and there is a lack of gene resources that can be used to improve plant salt tolerance and drought tolerance during transgenic molecular breeding. Summary of the Invention

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

[0005] To achieve the above object, the present invention provides the following technical solutions: The present invention provides a Medicago falcata WRKY47 transcription factor, the amino acid sequence of which is as shown in SEQ ID NO: 1.

[0006] The present invention provides a gene encoding the Medicago falcata WRKY47 transcription factor.

[0007] The present invention provides a gene-derived product containing the gene, including an expression cassette containing the gene; The expression cassette further contains a promoter, and the nucleotide sequence of the promoter is as shown in SEQ ID NO: 3.

[0008] The present invention provides an application of the Medicago falcata WRKY47 transcription factor, the gene, or the gene-derived product in at least one of the following works: 1) Improving plant salt tolerance and / or drought tolerance; 2) Creating salt-tolerant and / or drought-tolerant plant varieties.

[0009] Preferably, the Medicago falcata WRKY47 transcription factor or the gene improves the salt tolerance and / or drought tolerance of plants through positive regulation.

[0010] 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 H 2 O 2 accumulation amount and / or the accumulation amount of O 2- and increasing the antioxidant enzyme activity of plants and reducing the malondialdehyde content of plants.

[0011] Preferably, the plants include Medicago plants or Arabidopsis plants.

[0012] 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.

[0013] 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: 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.

[0014] 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.

[0015] The present invention has the following advantages compared with the prior art: 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 the salt tolerance and drought tolerance of heterologously expressed MfWRKY47 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 strains.

[0016] The present invention provides a method for improving the salt tolerance and / or drought tolerance of plants, which transfers the gene or a derivative containing the gene into plants. In the examples of the present invention, MfWRKY47Arabidopsis thaliana was genetically transformed by the floral dip method, and the heterologous expression obtained MfWRKY47 The salt tolerance and / or drought resistance of the Arabidopsis thaliana is significantly improved compared with that of the wild-type Arabidopsis thaliana. Description of the drawings

[0017] Figure 1 It is a picture of the hydroponic seedlings of Medicago falcata; Figure 2 It is Medicago falcata MfWRKY47 Phylogenetic tree analysis diagram; Figure 3 It is Medicago falcata MfWRKY47 Tissue expression characteristic analysis diagram; Figure 4 It is Medicago falcata MfWRKY47 Expression pattern analysis results diagram of the response to salt and drought stresses; where A is drought stress; B is salt stress; Figure 5 It is Medicago falcata MfWRKY47 Results diagram of the promoter-driven GUS expression pattern, where A is CK, B is salt stress, C is drought stress, and D is the GUS activity under drought stress; Figure 6 It is the subcellular localization verification results diagram of MfWRKY47 in Medicago falcata, and the scale bar is 20μm; Figure 7 It is the transcriptional activation function verification results diagram of MfWRKY47 in Medicago falcata; Figure 8 It is the verification results diagram of the binding of MfWRKY47 in Medicago falcata to the W-box; Figure 9 It is the heterologous expression in Arabidopsis thaliana MfWRKY47 Verification results diagram of the expression level of the transgenic lines; Figure 10 It is the heterologous expression in Arabidopsis thaliana MfWRKY47 Verification results diagram of the salt tolerance and drought resistance of the transgenic lines, where A is the seed germination rate; B is the main root length; C is the fresh weight of the plants; Figure 11 It is the heterologous expression after drought stress treatment MfWRKY47 Analysis results diagram of the leaf water loss rate of the transgenic lines; Figure 12 It is the heterologous expression MfWRKY47 Verification results diagram of the chlorophyll content of the transgenic lines; Figure 13 It is the heterologous expression MfWRKY47 The transgenic lines of H 2 O 2 Content determination and DAB staining results diagram; Figure 14 It is the heterologous expression MfWRKY47 The transgenic lines of O 2-Graphs of content determination and NBT staining results; Figure 15 Graphs of the activities of antioxidant enzymes (SOD), peroxidase (POD), and catalase (CAT) and the content of malondialdehyde (MDA) in heterologously expressed plants under drought stress; MfWRKY47 Graphs of the activities of antioxidant enzymes (SOD), peroxidase (POD), and catalase (CAT) and the content of malondialdehyde (MDA) in heterologously expressed plants under drought stress; Figure 16 Graphs of the activities of antioxidant enzymes (SOD), peroxidase (POD), and catalase (CAT) and the content of malondialdehyde (MDA) in heterologously expressed plants under salt stress; MfWRKY47 Graphs of the activities of antioxidant enzymes (SOD), peroxidase (POD), and catalase (CAT) and the content of malondialdehyde (MDA) in heterologously expressed plants under salt stress; Figure 17 Graphs of the results of regulating the expression of related genes in heterologously expressed plants under drought stress; MfWRKY47 Graphs of the results of regulating the expression of related genes in heterologously expressed plants under drought stress; Figure 18 Graphs of the results of regulating the expression of related genes in heterologously expressed plants under salt stress; MfWRKY47 Graphs of the results of regulating the expression of related genes in heterologously expressed plants under salt stress. Detailed implementation methods

[0018] The present invention provides a Medicago falcata WRKY47 transcription factor, the amino acid sequence of which is shown in SEQ ID NO: 1 (MLNLVNDRCNVLQNRLLLAMHMHQSSSLPQNNHNLLLKGNTQDAEKPVLPTRQFFDEPSPSDCSKNNGFAIVENNENNMGRNLACEYINEGEINSKIEDQSSEVGCRRARVSIRARSDFAFMVDGCQWRKYGQKTAKGNPCPRAYYRCSMGTSCPVRKQVQRCFKDESVFITTYEGNHNHQLPPAAKPIANLTSSALNTFLPTSSTNLQQYGNNLTNTFLFSSPLSPPNSNAIATFSPSPTCPTITLDFTLPPSNYLQFKNHKQSSLLPFPFQGHYPPSFEVFPNLINNERKLDLVDVVSEALEKDPSLKEALFSAMSSFTNGDSSNINNQSQLPSKSSG).

[0019] 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.

[0020] The present invention provides a gene encoding the Medicago falcata WRKY47 transcription factor.

[0021] 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, modification of the nucleotide sequence of the gene without changing the amino acid sequence also falls within the protection scope of the present invention.

[0022] The expression level of the gene of the present invention is positively correlated with plant salt tolerance and / or drought tolerance. Examples of the present invention show that MfWRKY47 it is expressed in the roots, stems and leaves of Medicago falcata, but has the highest expression in the roots. At the same time, MfWRKY47 the expression level can respond to changes in salt concentration and drought environment in the growth environment, and the expression level MfWRKY47 increases in a salt environment or a 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, under salt stress or drought stress conditions, the seeds of Arabidopsis thaliana plants expressing MfWRKY47 have a higher germination rate compared to those of wild-type plants, and there are also improvements in root length, fresh weight, etc., and the chlorophyll content is also increased, while the accumulation of H 2 O 2 and O 2- is reduced, and the antioxidant enzyme activity is increased. In addition, the malondialdehyde content is reduced, indicating that the stress resistance of Arabidopsis thaliana plants expressing MfWRKY47 is improved. At the same time, differentially expressed genes in heterologous expression MfWRKY47 Arabidopsis thaliana plants are analyzed. The results show that under drought stress, heterologous expression MfWRKY47 can significantly up-regulate the expression of stress response genes LEA4-5 , peroxidase genes PRX52 , and cation transporter genes CAX3 . Under salt stress, heterologous expression MfWRKY47 can significantly up-regulate the expression of nitrogen metabolism regulatory genes NIA2 , Na + / H + transporter genes NHX4 , and salt stress response genes CHX16 . It shows that MfWRKY47 the gene regulates the expression of stress-related genes, antioxidant enzyme genes, and ion transporter genes to enhance the salt tolerance and drought tolerance of plants.

[0023] The present invention provides a gene-derived product containing the gene, including an expression cassette containing the gene; the expression cassette further contains a promoter, and the nucleotide sequence of the promoter is as shown in SEQ ID NO: 3.

[0024] 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 arid 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, prepare salt-tolerant and / or drought-resistant transgenic plants, and be used in one or more of plant genetic breeding or germplasm improvement.

[0025] The present invention provides an application of the Medicago falcata WRKY47 transcription factor, the gene or the gene-derived product in at least one of the following tasks: 1) improving the salt tolerance and / or drought resistance of plants; 2) creating salt-tolerant and / or drought-resistant plant varieties.

[0026] In the present invention, the Medicago falcata WRKY47 transcription factor or the gene preferably improves the salt tolerance and / or drought resistance of plants through positive regulation. The salt tolerance preferably includes the resistance of plants to salt environment. 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 resistance preferably includes the resistance of plants to arid environment. The arid environment is preferably an arid environment simulated by mannitol, and the concentration of mannitol is preferably above 100 mM, more preferably 150 - 300 mM, and most preferably 200 mM.

[0027] In the present invention, the improvement of the salt tolerance and / or drought resistance 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 amount of H 2 O 2 and / or the accumulation amount of O 2- and increasing the antioxidant enzyme activity of plants and reducing the malondialdehyde content of plants.

[0028] 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, examples of the present invention verified in Arabidopsis thaliana that the Medicago falcata WRKY47 transcription factor or the gene has the effect of improving the salt tolerance and / or drought resistance of plants, and it can be obtained that within the range of dicotyledonous plants, it has the biological effect of improving the salt tolerance and / or drought resistance of plants.

[0029] The present invention provides a method for improving the salt tolerance and / or drought resistance of plants, which is to overexpress the Medicago falcata WRKY47 transcription factor or the gene-derived product in plants.

[0030] In the present invention, the derivative products containing the gene 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 include Agrobacterium tumefaciens GV3101. The present invention does not limit the method for transferring into plants, and the conventional methods for transforming plants in the art can be used. The plants are preferably the same as those in the above applications and will not be elaborated here. In the examples of the present invention, MfWRKY47 Arabidopsis thaliana was genetically transformed by the floral dip method, and the obtained heterologous expression MfWRKY47 Arabidopsis thaliana has significantly improved salt tolerance and / or drought tolerance compared with wild-type Arabidopsis thaliana.

[0031] The present invention provides a method for identifying the salt tolerance and / or drought tolerance of plants, detecting the expression level of the Medicago falcata WRKY47 transcription factor or the gene in the plants, and judging the salt tolerance and / or drought tolerance of the 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 tolerance of the plants.

[0032] In the present invention, the reagents for detecting the expression level of the gene preferably include the forward primer shown in SEQ ID NO: 4 and the reverse primer shown in SEQ ID NO: 5.

[0033] In the examples of the present invention, MfWRKY47 The heterologous expression MfWRKY47 Arabidopsis thaliana obtained by transforming Arabidopsis thaliana was detected by the above reagents, indicating that the heterologous expression MfWRKY47 in Arabidopsis thaliana MfWRKY47 has a high expression level ， and has significantly improved salt tolerance and / or drought tolerance compared with wild-type Arabidopsis thaliana. Therefore, the salt tolerance and / or drought tolerance of plants can be judged according to the expression level of the Medicago falcata WRKY47 transcription factor or the gene in the plants.

[0034] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below in conjunction with the drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0035] 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 sites.

[0036] Table 1 Primer names and nucleotide sequence information

[0037] Example 1 Obtaining the Medicago falcata MfWRKY47 transcription factor 1. Obtaining hydroponic seedlings of Medicago falcata 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 , 16 h light / 8 h dark) culture, and after 1 W, the seedlings with the same growth were selected and transplanted into a culture box filled with 1 / 2 Hoagland nutrient solution and cultured in a greenhouse (25±1℃, 16 h light / 8 h dark) to obtain hydroponic seedlings such as Figure 1 shown.

[0038] 2. Acquisition of MfWRKY47 transcription factor from Medicago truncatula Four-week-old alfalfa hydroponic seedlings were used as materials, leaves were collected, ground with liquid nitrogen, and total RNA was extracted using Trizol reagent (purchased from TaKaRa). Then, cDNA was synthesized by reverse transcription using a reverse transcription kit (purchased from TaKaRa, item number 6210A). Using cDNA as a template, primers MfWRKY47 -R and MfWRKY47 -F was used for RT-PCR amplification, and the amplification conditions were 98 ℃ for 5 min; 95 ℃ for 1 min, 56 ℃ for 30 s, 68 ℃ for 1 min, 30 cycles; 68 ℃ for 10 min. After the PCR reaction, the target fragment was purified using a PCR product purification kit (purchased from Sangon Biotechnology Co., Ltd.) and then ligated with pMD19-T to construct pMD -MfWRKY47 After ligation reaction at 16 °C for 3 h, the cells were transformed into competent cells Trans1-T1 and identified by bacterial PCR (the primers for bacterial PCR were MfWRKY47 -R and MfWRKY47 -F) and enzyme digestion verification, and sequencing analysis MfWRKY47 Transcription factor gene CDS sequence. 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.

[0039] The maximum likelihood method of MEGA software was used to construct the phylogenetic tree, and the Bootstrap value was set to 1000. Figure 2 Shown: MfWRKY47 has the highest homology with AtWRKY47.

[0040] Example 2 Yellow clover MfWRKY47Obtaining the Sequence of Gene Promoter MfWRKY47p Using the 4-week-old hydroponic seedlings of Medicago falcata in Example 1 as materials, collect the leaves. After grinding with liquid nitrogen, extract the genomic DNA of Medicago falcata by the CTAB method, and then construct a GenomeWalker DNA library using the GenomeWalker kit (TaKaRa). According to the obtained MfWRKY47 gene CDS nucleotides, design WRKY47 p-GSP1 and WRKY47 p-GSP2 primers. Using the GenomeWalker DNA library as a template, perform 2 rounds of PCR amplification. The conditions for the first round of PCR are: 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 are: 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 purifying the PCR products, ligate them with pMD19-T and transform them into Trans1-T1, and then perform sequencing analysis to obtain the DNA sequence. According to the obtained DNA sequence, design WRKY47 p-GSP3 and WRKY47 p-GSP4 primers. Using the GenomeWalker DNA library as a template, perform the second-step PCR walking amplification. After purifying the PCR products, ligate them with pMD19-T and transform them into Trans1-T1, and then perform sequencing analysis to obtain the DNA sequence. The DNA sequences obtained by the secondary walking amplification are spliced to obtain the promoter sequence DNA information. According to the obtained promoter sequence DNA information, design specific primers WRKY47p-R and WRKY47p-F, and perform PCR amplification using the genomic DNA of Medicago falcata as a template. The PCR amplification conditions are 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 purifying the PCR products, ligate them with pMD19-T to form the recombinant plasmid pMD-WRKY47p, and transform it into Trans1-T1, and then perform sequencing analysis to obtain the WRKY47p DNA sequence.

[0041] Medicago falcata MfWRKY47 The nucleotide sequence of the gene promoter MfWRKY47p of Medicago falcata is shown in SEQ ID NO: 3. Using PlantCARE (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) for prediction and analysis, the results show that it contains cis-acting elements such as response to drought stress and osmotic stress.

[0042] Example 3 Medicago falcata MfWRKY47 Analysis of gene expression pattern 1. Analysis of tissue expression characteristics pattern Using the 4-week-old hydroponic seedlings of Medicago falcata 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- The MfEFlα detected by qF primers was used as the internal reference gene.

[0043] 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.

[0044] 2. Analysis of expression patterns in response to salt and drought stresses Taking the 4-week-old hydroponic seedlings of Medicago falcata in Example 1, they were divided into a salt stress treatment group and a drought stress treatment group. The salt stress treatment group was to culture the 4-week-old hydroponic seedlings of Medicago falcata in Hoaglands nutrient solution containing 200 mM NaCl. The drought stress treatment group was to culture the 4-week-old hydroponic seedlings of Medicago falcata in Hoaglands nutrient solution containing 200 mM mannitol.

[0045] 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- The MfEFlα detected by qF primers was used as the internal reference gene.

[0046] 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.

[0047] 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.

[0048] 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 of [plant name] in roots, stems and leaves is induced by drought stress.

[0049] 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 of [plant name] in roots, stems and leaves is induced by salt stress.

[0050] 3. [plant name] MfWRKY47 Analysis of the expression pattern driven by the WRKY47p promoter of the [gene name] gene Construction of the expression vector and infiltration of Arabidopsis thaliana: The vector pORE R1 STOCK: CD3-929 was purchased from the Arabidopsis Biological Resource Center (ABRC). The vectors 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.

[0051] GUS staining and GUS activity analysis: 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.

[0052] 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.

[0053] Example 4 Yellow clover MfWRKY47 Subcellular localization analysis of gene-encoded proteins 1. Vector construction and identification methods 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 and KpnⅠ 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.

[0054] 2. Transient expression in Nicotiana benthamiana leaves 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). 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 MgCl 2 , 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. Inject the cultured bacterial liquid into the lower epidermis of tobacco leaves and culture in weak light for 48 h.

[0055] 3. Observation with a laser confocal microscope Under light-avoiding conditions, cut the tobacco leaves and place them in the DAPI (5 μg / mL) staining solution. After 15 min, rinse with 1×PBS buffer twice. Then place the leaves face up on a glass slide for preparation, 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.

[0056] The results are as Figure 6 shown: The protein encoded by the Medicago falcata MfWRKY47 gene is localized in the nucleus.

[0057] Example 5 Verification of the transcriptional activation function of the protein encoded by the Medicago falcata MfWRKY47 gene 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. 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 reached 0.5, and then diluted 10 -1 , 10 -2 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 transformation of pGBKT7-53 + pGADT7-T bacteria was used as a positive control, and the transformation of pGBKT7-Lam + pGADT7-T bacteria and the transformation of pGBKT7 bacteria were used as negative controls.

[0058] The results were as Figure 7 shown: The transcription activation function of the protein encoded by the Medicago falcata gene was not detected in yeast cells. MfWRKY47

[0059] Example 6 Verification experiment of the binding of the protein encoded by the Medicago falcata gene to the W-box MfWRKY47 1. Construction of the bait vector 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 pAbAi vector (purchased from Takara) digested with Sac I and Sal I, and the bait vectors pAbAi-W-box and pAbAi-mW-box were obtained after transformation into Trans1-T1 and sequencing analysis.

[0060] 2. Construction of the expression vector Design containing​​EcoR Ⅰ. Sac Ⅰ restriction site primers pAD-MfWRKY47-F and pAD-MfWRKY47-R were used to perform PCR amplification of the target fragment with the recombinant plasmid pMD in Example 1 as the template. The amplification reaction conditions were all: 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 -MfWRKY47 Ⅰ and purification of the PCR product, and the pGADT7 vector (purchased from Takara) were digested, and then 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 bacterial colony PCR and digestion, the positive plasmid vector was obtained. EcoR Ⅰ. Sac Ⅰ, the digested and purified PCR product and the pGADT7 vector (purchased from Takara) were ligated with T4 DNA Ligase at 16 °C for 12 h to construct the pGADT7-MfWRKY47 vector, which was introduced into Trans1-T1. After verification by bacterial colony PCR and digestion, the positive plasmid vector was obtained.

[0061] 3. Verification of binding characteristics 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 bacterial colony PCR using the primers W-box, pAbAi-R and mW-box, pAbAi-R in Table 1 respectively, the positive strains were obtained and denoted as W-Y1HGold and mW-Y1HGold respectively. The bacterial 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.

[0062] The pGADT7 and pGADT7-MfWRKY47 plasmids were respectively transformed into the bacterial 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 bacterial solution was adjusted to OD 600 = 0.5 with sterile 0.9% NaCl solution, and then diluted 10 -1 , 10 -2 and 10 -3 times in sequence. 15 μL of each diluted bacterial solution was taken and dropped on SD / Ura-Leu- / AbA (350 ng / mL) medium, and cultured at 30 °C for 3 d to observe the colony growth status.

[0063] The results are asFigure 8 Shown: Yellow clover MfWRKY47 The gene encodes a protein that binds to the W-box.

[0064] Example 7 Yellow clover MfWRKY47 Application analysis of gene regulation of plant salt tolerance and drought resistance 1. Heterologous Expression MfWRKY47 Method for creating transgenic lines of Arabidopsis thaliana Arabidopsis thaliana was used as the test plant, and the Bam HI Sac Primer pPZP221- MfWRKY47 -F and pPZP221- MfWRKY47 -R, with the recombinant plasmid pMD in Example 1 -MfWRKY47 The target fragment was amplified by PCR as a template. The amplification reaction conditions were: 98 ℃ for 5 min; 95 ℃ for 1 min, 57 ℃ for 30 s, 72 ℃ for 1 min, 30 cycles; 68 ℃ for 10 min. The PCR product was obtained after the amplification. Bam HI Sac The purified PCR product and pPZP221 (35s-nos) vector were digested by I and ligated with T4 DNA Ligase at 16 °C for 12 h to construct the heterologous expression vector pPZP221 (35s- MfWRKY47 - nos) was introduced into Trans1-T1, and a positive plasmid vector was obtained after bacterial PCR and restriction digestion verification. The heterologous expression vector pPZP221 (35s- MfWRKY47 - nos) was introduced into Agrobacterium GV3101. Arabidopsis was genetically transformed by inflorescence infiltration method, and the homozygous transgenic Arabidopsis T3 generation strains were obtained by screening and culturing with 50 mg / L Gen. The pPZP221 (35s-nos) vector is consistent with the pPZP221 (35s-nos) vector in the prior art (Niu Yiding. Research on DREB transcription factor genes of alfalfa [D]. Inner Mongolia University, 2008.).

[0065] RNA was extracted from fresh leaves of the homozygous transgenic Arabidopsis thaliana T3 strain, and the cDNA obtained after reverse transcription was used as a template. WRKY47 -qR and WRKY47 -qF primers were used to perform real-time PCR analysis using a fluorescent quantitative kit (TB Green™ Premix Ex Taq™ II, TaKaRa). Atactin2 is the internal reference gene.

[0066] The real-time PCR detection system was 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.

[0067] The reaction conditions were: 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.

[0068] The results were as Figure 9 shown: The MfWRKY47 gene was highly expressed in all transgenic lines. The gene expression levels in OE-8 transgenic line, OE-9 transgenic line, and OE-12 transgenic line MfWRKY47 were higher than those in 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.

[0069] 2. Verification experiments on salt tolerance and drought resistance of transgenic plants 1) Analysis of seed germination rate The seeds were divided into a salt stress treatment group and a drought stress treatment group. In the drought stress treatment group, the T3-generation seeds of wild type and transgenic lines were sown in 1 / 2 MS medium containing Mannitol (100 mM, 200 mM, and 300 mM) respectively; in the salt stress treatment group, the T3-generation seeds of wild type and transgenic lines were sown 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. All experiments were designed with 3 biological replicates and 3 technical replicates.

[0070] 2) Analysis of seedling growth indexes 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.

[0071] 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.

[0072] 3) Analysis of physiological and biochemical indexes of salt tolerance and drought resistance The T3 generation seeds of wild-type and transgenic lines were sown in 1 / 2 MS medium. After 14 days of cultivation, they were transferred to nutrient soil and routinely 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, H 2 O 2 and O 2- concentrations, 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.

[0073] 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 13 The results showed that H2 O 2 The accumulation amount was significantly reduced compared with the wild type. Figure 14 The results showed that the O accumulation amount in the transgenic lines was significantly reduced compared with the wild type after salt stress treatment or drought stress treatment. 2- The accumulation amount was significantly reduced compared with the wild type. Figure 15 The results showed that the activities of SOD, POD and CAT in the transgenic lines were significantly increased compared with the wild type plants, and the content of malondialdehyde (MDA) was significantly reduced after drought stress treatment. Figure 16 The results showed that the activities of SOD, POD and CAT in the transgenic lines were significantly increased compared with the wild type plants, and the content of malondialdehyde (MDA) was significantly reduced after salt stress treatment.

[0074] The above results showed that Medicago falcata MfWRKY47 genes can 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 H 2 O 2 and O 2- ( Figure 13 and Figure 14 ), increasing the antioxidant enzyme activity and reducing the degree of membrane damage ( Figure 15 and Figure 16 ).

[0075] 3. Medicago falcata MfWRKY47 Analysis of the regulation of salt tolerance and drought resistance related gene expression by Medicago falcata genes RNA-seq analysis of transgenic lines (OE-12 transgenic lines, denoted as OE in Figure 17 and Figure 18 ). After analyzing the differentially expressed genes by DEG analysis, AtActin1 was used as the internal reference gene for RT-qPCR verification, 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 , and cation transporter genes CAX3 in the transgenic lines were significantly increased compared with the wild type. Therefore, heterologous expression of MfWRKY47 under drought stress can significantly up-regulate the expression of stress response genes LEA4-5 , peroxidase genes PRX52 , and 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 can significantly upregulate the nitrogen metabolism regulatory genes 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.

[0076] 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 these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

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

1.

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

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

3.

4. Use of the WRKY47 transcription factor of Medicago truncatula according to claim 1, the gene according to claim 2 or the gene-derived product according to claim 3 in at least one of the following tasks: 1) Improve the salt tolerance and / or drought resistance of plants; 2) Create salt-tolerant and / or drought-resistant plant varieties.

5. The application according to claim 4, characterized in that: The alfalfa WRKY47 transcription factor or the gene improves the salt tolerance and / or drought resistance of the plant through positive regulation.

6. The use according to claim 4, characterized in that: The improvement of the salt tolerance and / or drought resistance of plants includes the change of at least one of the following indicators: increasing plant biomass, increasing plant chlorophyll content, reducing plant H2O2 accumulation and / or O 2- accumulation, increase the antioxidant enzyme activity of plants and reduce the malondialdehyde content of plants.

7. The use according to any one of claims 4 to 6, characterized in that: The plant includes a plant of the genus Medicago or a plant of the genus Arabidopsis.

8. A method for improving the salt tolerance and / or drought resistance of plants, characterized in that: Overexpressing the alfalfa WRKY47 transcription factor of claim 1, the gene of claim 2 or the gene derivative product of claim 3 in a plant.

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

10. The method according to claim 9, characterized in that: The reagent for detecting the expression level of the gene includes a forward primer shown in SEQ ID NO:4 and a reverse primer shown in SEQ ID NO:5.

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