Application of overexpressed soybean GmMYB306 gene in preparation of product for improving resistance of crops to soybean phytophthora root rot

Abstract

The invention relates to application of an overexpressed soybean GmMYB306 gene in preparation of a product for improving resistance of crops to soybean phytophthora root rot, and belongs to the technical field of bioengineering. The invention provides application of an overexpressed soybean GmMYB306 gene in preparation of a product for improving the resistance of crops to soybean phytophthora root rot. The nucleotide sequence of the soybean GmMYB306 gene is shown as SEQ ID NO. 1. The soybean GmMYB306 protein disclosed by the invention can interact with a disease-resistant related protein GmNPR1, and the defense capability of soybean plants to phytophthora sojae is improved by regulating and controlling the expression of a crop disease course related gene GmPR1, so that the soybean GmMYB306 protein plays an important role in improving the resistance of the soybean plants to phytophthora sojae root rot.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to application of overexpressed soybean GmMYB306 gene in preparing a product for improving crop resistance to soybean Phytophthora root rot. Background Art

[0002] Phytophthora root rot of soybeans is a devastating disease caused by the fungus Phytophthora sojae, which rots the soybean root and stem. The fungus is highly variable, and with the increase in new strains and the diversification of virulence, existing resistant soybean varieties can easily lose their resistance to the pathogen, posing a serious threat to soybean production.

[0003] MYB transcription factors are a diverse class of transcription factors found in plants. The first reported plant MYB transcription factor was the maize C1 gene, which is primarily responsible for regulating anthocyanin synthesis. Most MYB proteins contain a MYB domain at their N-termini. Based on the number of MYB domains, MYB transcription factors can be classified as 1R-MYB, 2R-MYB, 3R-MYB, and 4R-MYB. MYB transcription factors are widely involved in plant physiological activities, including growth and development and responses to biotic stresses, and play a vital role in these processes. Arabidopsis AtMYB44 regulates plant resistance to green peach aphid and diamondback moth through EIN2; Brassica juncea BjMYB1 activates the expression of BjCHI1 by specifically binding to the W-box-like-4 element of the BJC-P promoter, thereby improving the immunity of transgenic Arabidopsis plants to gray mold; rapeseed BnaMYB78 activates plant disease resistance by regulating ROS accumulation and the transcription of defense-related genes; GsMYB15 identified from the soybean variety "ED059" is induced by NaCl, MeJA, SA and insect attack, and overexpression of GsMYB15 can enhance the plant's resistance to cotton bollworm larvae; apple MdMYB30 binds to the MdKCS1 promoter to activate and regulate wax biosynthesis, thereby improving the plant's resistance to Pseudomonas syringae pv. tomato Pst DC3000 and ring rot pathogen; silencing CaPHL8 can directly lead to reduced immunity of pepper to bacterial wilt pathogen and reduced expression of defense-related marker genes, while transient overexpression of CaPHL8 can cause allergic reactions in pepper and H2O2 and increase the expression of immunity-related marker genes; wild grape VdMYB1 binds to the MYBBS binding site in the promoter of the stilbene synthase gene STS2 to activate the transcription of STS2, which can promote the accumulation of resveratrol in grape leaves and improve the plant's immunity to powdery mildew; when rice is attacked by brown planthoppers, OsMYB30 can upregulate OsPAL6 and OsPAL 8; in apple, MdMYB73 is strongly induced after inoculation with the fungus Botrytis cinerea, enhancing plant resistance to Botrytis cinerea through the SA pathway; in citrus, CsMYB96 enhances plant resistance to gray mold by activating SA biosynthesis and promoting the accumulation of defense metabolites; in cucumber, CsMYB60 enhances plant resistance to Fusarium solani by increasing proanthocyanidin biosynthesis; in tomato, SlMYBs participate in the tomato defense response to Sclerotinia sclerotiorum; and in chickpea, CaMYB39 blocks infection by Ascochyta rabiei by regulating the biosynthesis of flavonols, phenylpropanoids, and isoflavonoids, reducing reactive oxygen species, and inducing the expression of defense genes. Therefore, MYB transcription factors play an important role in plant-pathogen interactions.

[0004] However, existing technologies lack relevant research on the role of soybean MYB transcription factors in improving soybean plant disease resistance and preventing and controlling soybean Phytophthora root rot. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of overexpressing the soybean GmMYB306 gene in the preparation of a product for improving crop resistance to soybean Phytophthora root rot, so as to solve the problem that the existing technology lacks the application of soybean MYB transcription factors in improving crop disease resistance, preventing and controlling soybean Phytophthora root rot, etc.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an application of an overexpressed soybean GmMYB306 gene in preparing a product for improving crop resistance to soybean Phytophthora root rot. The nucleotide sequence of the soybean GmMYB306 gene is shown in SEQ ID NO.1.

[0008] The present invention also provides an application of an overexpressed soybean GmMYB306 gene in preparing a product for increasing the expression of crop disease-related genes, wherein the nucleotide sequence of the soybean GmMYB306 gene is shown in SEQ ID NO.1;

[0009] The crop disease course-related gene is the GmPR1 gene.

[0010] The present invention also provides an overexpression recombinant plasmid, which comprises the soybean GmMYB306 gene and an overexpression empty vector;

[0011] The nucleotide sequence of the soybean GmMYB306 gene is shown in SEQ ID NO.1;

[0012] The overexpression empty vector is pCAMBIA3301 vector.

[0013] The present invention also provides a method for constructing the overexpression recombinant plasmid, which comprises connecting the soybean GmMYB306 gene to an empty overexpression vector to obtain the overexpression recombinant plasmid.

[0014] The present invention also provides a primer pair, comprising an upstream primer and a downstream primer;

[0015] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.25;

[0016] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.26.

[0017] The present invention also provides application of the primer pair in detecting the expression level of soybean GmMYB306 gene.

[0018] The present invention also provides a recombinant bacterium, which comprises the overexpression recombinant plasmid and an empty vector bacterium;

[0019] The empty-loaded bacteria are Escherichia coli Trans1-T1 strains.

[0020] The present invention also provides the use of the overexpression recombinant plasmid or the recombinant bacteria in improving the resistance of crops to soybean Phytophthora root rot.

[0021] The present invention also provides the use of the overexpression recombinant plasmid or the recombinant bacteria in increasing the expression level of crop disease-related genes.

[0022] The present invention has the following technical effects and advantages:

[0023] The GmMYB306 of the present invention can interact with the disease resistance-related protein GmNPR1 and improve the defense ability of soybean plants against soybean Phytophthora by regulating the expression of the crop disease process-related gene GmPR1, thereby playing an important role in improving the resistance of soybean plants to soybean Phytophthora root rot. It provides an important genetic basis and theoretical support for studying the relevant mechanisms of resistance to soybean Phytophthora root rot, provides valuable genetic resources for promoting the research and application of plant defense systems and cultivating new soybean varieties with high disease resistance, and has important application value in soybean disease-resistant genetic engineering breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the domain prediction result of GmMYB306 protein;

[0025] Figure 2 For analysis of the autoactivation activity of GmNPR1;

[0026] Figure 3 This is the result of 3-AT inhibiting the autoactivation of GmNPR1;

[0027] Figure 4 The yeast two-hybrid validation results for GmMYB306 and GmNPR1;

[0028] Figure 5 The results of LUC interaction validation between GmMYB306 and GmNPR1 are shown;

[0029] Figure 6Figure 1 is the SDS-PAGE electrophoresis result of the recombinant protein GmMYB306-His. Lane 1 is the supernatant obtained by ultrasonic lysis of the bacteria after IPTG induction, lane 2 is the precipitate obtained by ultrasonic lysis of the bacteria after IPTG induction, lane 3 is the flow-through, lane 4 is the recombinant protein GmMYB306-His after IPTG induction at 37°C for 4 hours, and lanes 5 to 9 are the eluates from the first to fifth purification recovery of the recombinant protein GmMYB306-His, respectively.

[0030] Figure 7 The figures are the results of SDS-PAGE electrophoresis and Western blot detection of the recombinant protein GmNPR1-GST. The left figure shows the SDS-PAGE electrophoresis result of the recombinant protein GmNPR1-GST, and lanes 1 to 5 in the figure are the eluates from the first to fifth purification and recovery of the recombinant protein GmNPR1-GST, respectively. The right figure shows the results of Western blot detection of the recombinant protein GmNPR1-GST, and lanes 6 to 10 in the figure are the eluates from the first to fifth purification and recovery of the recombinant protein GmNPR1-GST, respectively.

[0031] Figure 8 The results of the in vitro pull-down interaction between GmMYB306 and GmNPR1 were verified;

[0032] Figure 9 The activation result of GmPR1 by GmMYB306;

[0033] Figure 10 This is the regulatory result of GmMYB306 on GmPR1;

[0034] Figure 11 The fluorescence observation results of GFP protein in the hairy roots of soybean plants of various strains;

[0035] Figure 12 The results of exogenous bar gene detection in hairy roots of soybean plants of various strains;

[0036] Figure 13 The expression level of GmMYB306 in the hairy roots of soybean plants of various strains is detected;

[0037] Figure 14 The following are the observation results of hairy root disease symptoms of soybean plants of various strains;

[0038] Figure 15 The expression levels of GmTEF1 and GmPSPEL1 in soybean plants of various lines were detected. DETAILED DESCRIPTION

[0039] The present invention provides an application of an overexpressed soybean GmMYB306 gene in preparing a product for improving crop resistance to soybean Phytophthora root rot. The nucleotide sequence of the soybean GmMYB306 gene is shown in SEQ ID NO.1.

[0040] The present invention also provides an application of an overexpressed soybean GmMYB306 gene in preparing a product for increasing the expression of crop disease-related genes, wherein the nucleotide sequence of the soybean GmMYB306 gene is shown in SEQ ID NO.1;

[0041] The crop disease course-related gene is the GmPR1 gene.

[0042] The present invention also provides an overexpression recombinant plasmid GmMYB306-Myc, wherein the overexpression recombinant plasmid GmMYB306-Myc comprises a soybean GmMYB306 gene and an overexpression empty vector;

[0043] The nucleotide sequence of the soybean GmMYB306 gene is shown in SEQ ID NO.1;

[0044] The overexpression empty vector is pCAMBIA3301 vector, which comes from the Key Laboratory of Soybean Biology, Ministry of Education, Northeast Agricultural University.

[0045] The present invention also provides a method for constructing the overexpression recombinant plasmid GmMYB306-Myc, which comprises connecting the soybean GmMYB306 gene to an empty overexpression vector to obtain the overexpression recombinant plasmid GmMYB306-Myc.

[0046] The present invention also provides a primer pair, comprising an upstream primer and a downstream primer;

[0047] The upstream primer is GmMYB306-QF, and its nucleotide sequence is shown in SEQ ID NO.25;

[0048] The downstream primer is GmMYB306-QR, and its nucleotide sequence is shown in SEQ ID NO.26.

[0049] The present invention also provides application of the primer pair in detecting the expression level of soybean GmMYB306 gene.

[0050] The present invention also provides a recombinant bacterium, which comprises the overexpression recombinant plasmid GmMYB306-Myc and an empty vector bacterium;

[0051] The empty vector bacteria are Escherichia coli Trans1-T1 strains, purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0052] The present invention also provides the use of the overexpression recombinant plasmid GmMYB306-Myc or the recombinant bacteria in improving the resistance of crops to soybean Phytophthora root rot.

[0053] The present invention also provides the use of the overexpression recombinant plasmid GmMYB306-Myc or the recombinant bacteria in increasing the expression level of crop disease-related genes;

[0054] The crop disease course-related gene is the GmPR1 gene.

[0055] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Among the vectors and plasmids of the present invention, the pEasy-Blunt vector was purchased from Shanghai Novozymes Biotechnology Co., Ltd., the pGreenⅡ0800 vector and the LUC empty effector vector were purchased from Wuhan Miaoling Biotechnology Co., Ltd., and the pGADT7 vector, pGBKT7 vector, pCAMBIA1300-nLuc vector, pCAMBIA1300-cLuc vector, pET-29b(+) vector, pGEX-4T-1 vector, pCAMBIA3301 vector, pFGC5941 vector, pGBKT7-53 plasmid, pGADT7-SV40 plasmid, Fls2-nLuc plasmid, and Gβ-cLuc plasmid were from the Key Laboratory of Soybean Biology, Ministry of Education, Northeast Agricultural University;

[0057] Among the strains of the present invention, Escherichia coli Trans1-T1 competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd., Escherichia coli DH5α competent cells and Agrobacterium K599 competent cells were purchased from Shanghai Novozyme Biotechnology Co., Ltd., Agrobacterium GV3101 (pSoup-p19) competent cells, Agrobacterium GV3101 strain, yeast Y2H cells, yeast Y2H Gold cells, prokaryotic expression host SHuffle T7 strain, and prokaryotic expression host Rosetta (DE3) strain were purchased from Shanghai Weidi Biotechnology Co., Ltd., and Phytophthora sojae was obtained from the Key Laboratory of Soybean Biology, Ministry of Education, Northeast Agricultural University.

[0058] Among the test materials of the present invention, the highly resistant soybean variety "Suinong 10" and the soybean variety "Dongnong 50" were from the Key Laboratory of Soybean Biology, Ministry of Education, Northeast Agricultural University;

[0059] Among the reagents of the present invention, Trizol reagent and Omega plasmid mini-extraction kit were purchased from Omega Corporation of the United States, ReverTraAce qPCR RT Kit was purchased from TOYOBO Corporation of Japan, KOD MasterMix was purchased from Nanjing Novozymes Biotechnology Co., Ltd., bar protein rapid detection test strips were purchased from Shanghai Novozymes Biotechnology Co., Ltd., ClonExpressⅡOne Step Cloning Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd., Super yeast competent state preparation and transformation kit and 3-amino-1,2,4-triazole (3-AT) were purchased from Beijing Coolbo Technology Co., Ltd., DDO medium, QDO medium, and QDO+X-α-Gal medium were purchased from Takara Corporation of Japan, D-Luciferin solution was purchased from Nanjing Wobo Biotechnology Co., Ltd., GST protein was from the Key Laboratory of Soybean Biology, Ministry of Education, Northeast Agricultural University, and His protein and GST antibody were purchased from Abmart Pharmaceutical Technology (Shanghai) Co., Ltd.

[0060] Example 1: Acquisition of soybean GmMYB306 gene

[0061] (1) Primer design: Based on the soybean GmMYB306 gene (NCBI ID: XM003524764.5), primers for amplifying the nucleotide sequence of the GmMYB306 gene (as shown in SEQ ID NO. 1) were designed and commissioned to Beijing Ruibo Xingke Biotechnology Co., Ltd. for synthesis. The nucleotide sequences of the primers are shown in Table 1.

[0062] Table 1 Primer sequences for amplifying the GmMYB306 gene

[0063] name Sequence (5'~3') SEQ ID NO. GmMYB306-F CATATGATGGGGAGGCCACCTTG 3 GmMYB306-R GGATCCCTAAAACAAACCTGCGGTACT 4

[0064] The nucleotide sequence of the GmMYB306 gene is shown in SEQ ID NO.1.

[0065] SEQ ID NO.1:

[0066] ATGGGGAGGCCACCTTGCTGTGACAAAATTGGGATTAAGAAAGGACCTTGGACTCCAGAAGAAGACATCATCTTGGTGTCTTACATTCAAGAACAGGGACCAGGGAATTGGAGAGCAGTTCCAACCAACACAGGTTTGATGAGATGCAGCAAGAGCTGCAGGCTTAGATGGACTAACTATCTTCGACCAGGTATCAAACGAGGTAATTTCACGGAACATGAAGAGAAGATGATAATCCACCTCCAAGCCCTTTTGGGGAACAGATGGGCTGCAATAGCTTCATATCTTCCACAAAGGACAGACAACGACATAAAAAACTATTGGAACACCCATTTGAAGAAGAAGCTGAAACAAAGTGGGAGTGATGAGGGTGTTGACCAAGAGGGACATTCTTCTTCTTCTTCTTCTAATTCACATCCAAAGGGTCAATGGGAGAGAAGGCTACAAACAGATATCCAAATGGCCAAGAAAGCCTTGTGTGATGCTTTGTCCCTTCACAAGCCAGCAACAGCAACAGCAACAAACCTTGTTGTGCCTGATGATGCCACCAAACCTTCTTCAACTTCTCACCAA CCCTACAAGCATGCATCATCCTCATATGCATCAAGCTACGAGAACATTTCACGTTTGATGGAAAACTGGATGAAATCCCCAAACTCAAACTCAACAAATAATTCGCCAGGGTATTATTCTTCTTCCTTCAGCAACATGGTCAATAATAACACTACAACTGGATCCAGTTCTAGTGAGGGAGCACATAGCAACACCACTACTACAACACAAGATCAGGGTTTTGACTCCTTGTTAACCCTAAACTCCTCCAAACACCATGGCTCTTCATCTCAGACGCAAGTGCCTCTTACTCTGCTGGAGAATTGGCTCTTTGATGATGGGGCTGCTCAGTGCCATGAAGATCTGATGAACATGTCGCTCGAAGAAAGTACCGCAGGTTTGTTTTAG

[0067] The amino acid sequence of the encoded GmMYB306 protein is shown in SEQ ID NO.2.

[0068] SEQ ID NO.2:

[0069] MGRPPCCDKIGIKKGPWTPEEDIILVSYIQEQGPGNWRAVPTNTGLMRCSKSCRLRWTNYLRPGIKRGNFTEHEEKMIIHLQALLGNRWAAIASYLPQRTDNDIKNYWNTHLKKKLKQSGSDEGVDQEGHSSSSSSNSHPKGQWERRLQTDIQMAKKAL CDALSLHKPATATATNLVVPDDATKPSSTSHQPYKHASSSYASSYENISRLMENWMKSPNSNSTNNSPGYYSSSFSNMVNNNTTTGSSSSEGAHSNTTTTTQDQGFDSLLTLNSSKHHGSSSQTQVPLTLLENWLFDDGAAQCHEDLMNMSLEESTAGLF

[0070] (2) Template acquisition: Leaves of soybean varieties 10 from Suinong were freeze-dried in liquid nitrogen, and soybean RNA was extracted using Trizol reagent. The specific steps were referred to the operating instructions of Trizol reagent. Then, soybean cDNA was obtained by reverse transcription using ReverTra Ace qPCR RT Kit. The specific steps were referred to the operating instructions of ReverTraAce qPCR RT Kit.

[0071] (3) PCR amplification: The amplification system was 50 μL, including 25 μL KOD MasterMix, 4 μL soybean cDNA, 1.5 μL GmMYB306-F primer, 1.5 μL GmMYB306-R primer, and 18 μL deionized water. The PCR amplification conditions were as follows: pre-denaturation at 98°C for 3 min, followed by denaturation at 98°C for 10 s → annealing at 60°C for 5 s → extension at 68°C for 5 s, for a total of 35 cycles, and finally extension at 68°C for 10 min. The GmMYB306 amplification product was obtained, and the domain structure prediction was performed using NCBI-Domain (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi). The results are as follows: Figure 1 shown.

[0072] The results showed that the amino acid sequence of GmMYB306 protein contained a PLN03212 domain, indicating that the GmMYB306 gene belongs to the MYB transcription factor.

[0073] Example 2: Vector construction and transformation

[0074] (1) Construction of cloning plasmid: Referring to the operating instructions of the pEasy-Blunt vector, the GmMYB306 amplified product was ligated into the pEasy-Blunt vector to obtain the cloning vector pEasy-Blunt-GmMYB306, which was then transformed into Escherichia coli DH5α competent cells and cultured. The cloning plasmid pEasy-Blunt-GmMYB306 was extracted using the Omega plasmid miniprep kit. For specific steps, refer to the operating instructions of the Omega plasmid miniprep kit.

[0075] (2) Amplification of target gene fragments: Referring to the operating instructions of the ClonExpressⅡOne Step Cloning Kit, specific primers with vector end sequences at both ends for amplification of each target gene were designed based on the GmMYB306 gene, GmNPR1 gene (NCBI ID: NM001251745.1), and GmPR1 gene (NCBI ID: NM001250324.3) using the primer design software CE Design (V1.04). The primers were then commissioned to Beijing Ruibo Xingke Biotechnology Co., Ltd. for synthesis. The nucleotide sequences of the specific primers are shown in Table 2. Using the soybean cDNA in Example 1 as a template, PCR amplification was performed to obtain the target gene fragments of GmNPR1 and GmPR1; using the cloning plasmid pEasy-Blunt-GmMYB306 as a template, PCR amplification was performed to obtain the target gene fragments of GmMYB306; wherein the PCR amplification system (50 μL) included 25 μL of KOD MasterMix, 4 μL of template, 1.5 μL each of upstream and downstream specific primers, and 18 μL of deionized water; the PCR amplification conditions were: pre-denaturation at 94°C for 3 min, followed by denaturation at 94°C for 30 s → annealing at 60°C for 30 s → extension at 72°C for 60 s, for a total of 30 cycles, and a final extension at 72°C for 10 min.

[0076] The nucleotide sequence of the NPR1 gene is shown in SEQ ID NO.5.

[0077] SEQ ID NO.5:

[0078] ATGGCTTATTCAGCCGAACCCTCATCATCTTTGAGCTTTACCTCATCTTCCCATCTATCAAATGGGTCGGTTAGTCACAACATATGCTCTTCTTATGGCTCTGACCCTGGACCTAACCTTGAGGCTCTCAGTTTGAGTAAGCTTAGCTCCAATTTTGAGCAGCTTTTGATTGAAACTGATTGTGATTATAGTGATGCTGACATCGTT GTGGAGGGAATTTCGGTTAGTGTTCATCGATGTATTCTGGCCTCTAGGAGTAAGTTTTTCCATGAATTATTCAAGAGAGAGAAGGGTTCATCAGAAAGAAGGGAAATTGAAGTATAACATGAGTGATTTGTTGCCTTATGGCAAGGTTGGATATGAAGCCTTCCTCATATTCCTTGGCTATGTATATACTGGTAAACTCAAGCCCT CTCCAATGGAAGTGTCTACTTGTGTTGACAGTGTGTGTGCCCATGATGCTTGTAGACCTGCCATTAACTTTGCTGTGGAGTTGATGTATGCCTCTTACATTTTTCAAATACCAGAGTTTGTATCACTTTTCCAGAGACGTCTACTTAACTTTATAGGGAAGGCTCTTGTGGAAGATGTCATCCCAATCCTCACTGTTGCTTTCCATTGTCAATTGAGTCAACTTGTCAATCAATGCATTGATAGGGTGGCCAGATCAGACCTTGACCAGATTTCAATTGACCAAGAGCTTCCAAATGAACTCTCACAAAAAGTTAAATTGCTCCGCCGCAACCCTCAGCGAGATGTTGAAAACGATGCTTCTATAGTGGATGCTTTGTCTCTAAAAAGAATCACTAGAATACACAAGGCATTGGATTCAGATGATGTTGAGCTTGTTAAACTTCTTTTAAATGAATCAGACATTACTTTAGATGAAGCCAATGCTCTCCATTATGCTGCAGCCTACTGTGATCCCAAGGTTGTTTCTGAGGTACTTGGTTTGGGACTCGCTAATGTCAATCTTCGAAATTCTAGGGGGTACACAGTGCTTCACATTGCTGCCATGCGTAAAGAGCCTTCCATTATAGTATCCCTACTTACGAAAGGGGCTTGTGCATCAGATTTGACTTTTGATGGTCAGAGTGCTGTTAGTATTTGTAGGAGGTTGACAAGGCCAAAGGATTATCATGCAAAAACAGAGCAGGGGAAAGAAACAAACAAAGATCGGATATGCATCGATGTTCTTGAAAGAGAAATGTGGAGGAATCCATTGGCTGGGGATGCCTGTATGTCTTCCCATACCATGGCTGATGATCTCCACATGAAACTACTATACCTTGAGAACAGAGTGGCATTTGCAAGACTTTTCTTCCCTTCAGAAGCCAAACTAGCCATGGACATTGCACATGCTGAGACAACATCTGAGTTTGCTGGTCTTTCTGCATCAAACTCAAAAGGTTCAAATGGAAACTTAAGGGAGGTTGATCTCAATGAAACTCCTATAGTTCAAAGTAAAAGACTTTTTTCTAGAATGGAAGCCCTTATGAAAACAGTGGAGATGGGGCGGCGCTACTTCCCGCATTGCTCGGAAGTGTTGGATAAGTTCATGGAGGATGACTTGCCTGACTTGTTTTACCTTGAAAAGGGTACTAATGAAGAGCAGAGAATCAAAAGGACACGTTTCATGGAGCTTAAAGATGACGTCCACAAGGCTTTCAACATGGACAAGGCCGAGTTTAGCCGCTCTGGGATTTCATCTTCATCATCCTCATCATCCCTCAGAGATTCTGTTGTACATTACAAGGCTAGGAAAGTGTAA

[0079] The nucleotide sequence of the PR1 gene is shown in SEQ ID NO.6.

[0080] SEQ ID NO.6

[0081] ATGATGTCCCCATCCCATGTGATCCTATCCATATTTTTCTTGGTGTGTACAACAACACCACCACTGTCCCTTGCCCAGAACACCCCTCAAGACTTTCTTGATGTGCACAATCAGGCTCGTGCCGAGGTTGGTGTTGGTCCACTCTCATGGAACCACCCCCTTCAAGCCTACGCTCAAAGGTATGCCAATGAGAGAATCCCTGACTGCAACCTCGAACACTCCATGGGACCCTTCGGCGAGAAT CTCGCTGAAGGGTACGGCGAAATGAAGGGTTCGGATGCTGTCAAATTTTGGCTCACTGAGAAGCCTTACTATGACCACTACTCCAACGCTTGTGTCCATGATGAGTGCTTGCATTATACTCAGATTGTGTGGCGTGATTCTGTTCATCTTGGGTGTGCTAGAGCAAAGTGTAACAATGGCTGGTGTTTGTTATTTGCAGCTATTCCCCACCAGGCAACATTGAAGGGGAACGACCTTATTGA

[0082] Table 2 Specific primer sequences for amplifying target gene fragments

[0083]

[0084]

[0085] (3) Linearization of empty vectors: pGreenⅡ0800 vector, pGADT7 vector, pGBKT7 vector, pCAMBIA1300-nLuc vector, pCAMBIA1300-cLuc vector, pET-29b(+) vector, pGEX-4T-1 vector, pCAMBIA3301 vector, and pFGC5941 vector were double-enzyme digested to obtain linearized empty vectors.

[0086] (4) Construction of recombinant plasmids: The target gene fragments of GmMYB306 obtained in step (2) were ligated into pGADT7 linearized empty vector, pCAMBIA1300-nLuc linearized empty vector, pET-29b(+) linearized empty vector, pCAMBIA3301 linearized empty vector, and pFGC5941 linearized empty vector, respectively, to obtain the corresponding recombinant vectors GmMYB306-AD, GmMYB306-nLuc, GmMYB306-His, GmMYB306-Myc, and GmMYB306-myc. YB306-RNAi; the target gene fragments of GmNPR1 obtained in step (2) were respectively connected to the pGBKT7 linearized empty vector, the pCAMBIA1300-cLuc linearized empty vector, and the pGEX-4T-1 linearized empty vector to obtain the corresponding recombinant vectors GmNPR1-BD, GmNPR1-cLuc, and GmNPR1-GST; the target gene fragment of GmPR1 obtained in step (2) was connected to the pGreenⅡ0800 linearized empty vector to obtain the recombinant vector GmPR1-LUC. Each recombinant vector was transformed into Escherichia coli Trans1-T1 competent cells and cultured. The recombinant plasmids were extracted using the Omega plasmid mini kit. The specific steps refer to the operating instructions of the Omega plasmid mini kit.

[0087] Example 3: Inhibition of GmNPR1 autoactivation

[0088] (1) Self-activation analysis: Yeast Y2H cells were prepared into yeast Y2H competent cells using the Super Yeast Competent Preparation and Transformation Kit. The recombinant plasmid GmNPR1-BD + pGADT7 vector, pGADT7 vector + pGBKT7 vector (negative control), and pGBKT7-53 plasmid + pGADT7-SV40 plasmid (positive control) were co-transformed into yeast Y2H competent cells, and the cells were analyzed by dot blot on DDO medium, QDO medium, and QDO + X-α-Gal medium. The results are as follows: Figure 2 shown.

[0089] (2) Inhibition of autoactivation: 3-AT is a competitive inhibitor of histidine and can inhibit the leaky expression of HIS3. The recombinant plasmid GmNPR1-BD + pGADT7 vector was co-transformed into yeast Y2H Gold cells and spread on DDO medium. It was inverted and cultured at 30°C for 3 days. The positive colonies were picked and expanded for 12 hours. Then, the cells were spotted on DDO medium, QDO medium, and QDO + X-α-Gal medium with 3-AT concentrations of 1 mmol / L and 2 mmol / L, respectively. The results are shown in Figure 2. Figure 3 shown.

[0090] The results of self-activation analysis showed that yeast co-transformed with the recombinant plasmid GmNPR1-BD + pGADT7 vector could grow normally in DDO medium, QDO medium, and QDO + X-α-Gal medium, indicating that GmNPR1 has self-activation activity;

[0091] Autoactivation inhibition results showed that yeast Y2H Gold cells co-transformed with the recombinant plasmid GmNPR1-BD + pGADT7 vector and the positive control could grow normally in QDO medium containing 1 mmol / L 3-AT and activated the expression of the reporter gene β-galactosidase, resulting in a blue color in QDO + X-α-gal medium. However, yeast Y2H Gold cells co-transformed with the recombinant plasmid GmNPR1-BD + pGADT7 vector failed to grow normally in QDO medium containing 2 mmol / L 3-AT or in QDO + X-α-gal medium, and failed to activate β-galactosidase expression. This indicates that the addition of 2 mmol / L 3-AT can inhibit the autoactivation effect of GmNPR1.

[0092] Example 4: Verification of protein interaction between GmMYB306 and GmNPR1

[0093] (1) Yeast two-hybrid verification: pGBKT7-53 plasmid + pGADT7-SV40 plasmid, recombinant plasmid GmMYB306-AD + recombinant plasmid GmNPR1-BD, GmBHLH68-AD plasmid + recombinant plasmid GmNPR1-BD, pGADT7 vector + pGBKT7 vector were co-transformed into yeast Y2H Gold cells, and then dot-blot analysis was performed on DDO medium, QDO medium, and QDO + X-α-gal medium containing 2 mmol / L 3-AT. The results are as follows: Figure 4 shown.

[0094] (2) LUC interaction verification: The recombinant plasmid GmMYB306-nLuc, recombinant plasmid GmNPR1-cLuc, pCAMBIA1300-nLuc vector, pCAMBIA1300-cLuc vector, Fls2-nLuc plasmid, and Gβ-cLuc plasmid were transformed into Agrobacterium GV3101 (pSoup-p19) competent cells, and the corresponding bacterial liquids were obtained. The recombinant plasmid GmMYB306-nLuc bacterial liquid + recombinant plasmid GmNPR1-cLuc bacterial liquid, ... Luc bacterial solution + pCAMBIA1300-cLuc vector bacterial solution (negative control), recombinant plasmid GmNPR1-cLuc bacterial solution + pCAMBIA1300-nLuc vector bacterial solution (negative control), Fls2-nLuc plasmid bacterial solution + Gβ-cLuc plasmid bacterial solution (positive control) were co-injected into Nicotiana benthamiana for transient expression. After 3 days of dark culture, 1 mmol / LD-Luciferin solution was sprayed and imaging was performed using the Tianneng 5200 chemiluminescence system. The results are shown in the figure. Figure 5 shown.

[0095] (3) Protein purification: The recombinant plasmid GmMYB306-His was transformed into the prokaryotic expression host SHuffle T7 strain and the strain was activated to OD 595nm = 0.5, 0.1 mmol / L IPTG was added and induced at 37 ° C for 4 h, and then the bacteria were ultrasonically lysed using the Biyuntian His protein purification kit, and the supernatant was subjected to SDS-PAGE electrophoresis to obtain the recombinant protein GmMYB306-His (such as Figure 6 Then the recombinant plasmid GmNPR1-GST was transformed into the prokaryotic expression host Rosetta (DE3) strain and the strain was activated to OD 595nm =0.5, 0.5mmol / L IPTG was added and induced at 37℃ for 6h, and then the cells were ultrasonically lysed using the Biyuntian GST protein purification kit. The supernatant was subjected to SDS-PAGE electrophoresis to obtain the recombinant protein GmNPR1-GST, and Western blot detection was performed (such as Figure 7 shown).

[0096] (4) In vitro pull-down interaction verification: 30 μL of Glutathione Sepharose 4B resin was transferred to an EP tube, centrifuged at 500 × g for 2 min, and the supernatant was removed; 1 × Binding Buffer was added to the EP tube, centrifuged at 500 × g for 2 min, and repeated once; GST protein and recombinant protein GmNPR1-GST were added to the EP tube respectively, and the mixture was combined at 4°C for 60 min, and then the recombinant protein GmMYB306-His was added respectively, and the mixture was combined at 4°C for 3 h, centrifuged at 500 × g for 2 min, and the supernatant was removed; 1 × Binding Buffer was added to the EP tube, washed at 4°C for 10 min, centrifuged at 500 × g for 2 min, and the washing was repeated 4 times; 40 μL of 5 × SDS loading buffer was added to the Glutathione Sepharose 4B resin, and the mixture was fully denatured in a 100°C water bath. His protein and GST antibody were added and Western blot detection was performed. The results are as follows: Figure 8 shown.

[0097] Yeast two-hybrid validation results showed that yeast Y2HGold cells co-transformed with pGBKT7-53 plasmid + pGADT7-SV40 plasmid, recombinant plasmid GmMYB306-AD + recombinant plasmid GmNPR1-BD, and GmBHLH68-AD plasmid + recombinant plasmid GmNPR1-BD, respectively, were able to grow normally in DDO medium and QDO medium, and activated β-galactosidase expression, thereby appearing blue in QDO + X-α-gal medium. This indicates that GmMYB306 and GmNPR1 can interact in yeast;

[0098] The LUC interaction validation results showed that chemiluminescent signals were detected in the leaves of Nicotiana benthamiana co-transfected with the positive control and the recombinant plasmid GmMYB306-nLuc solution plus the recombinant plasmid GmNPR1-cLuc solution, while no chemiluminescent signal was detected in the co-transfected negative control, indicating that GmMYB306 and GmNPR1 can interact in plants.

[0099] Protein purification results showed that both the recombinant protein GmMYB306-His and the recombinant protein GmNPR1-GST could be successfully purified and recovered;

[0100] The results of in vitro pull-down interaction verification showed that the recombinant proteins GmNPR1-GST, GmMYB306-His and GST proteins corresponding to the input lane and the recombinant proteins GmNPR1-GST and GST proteins corresponding to the pull-down lane were all expressed normally and bound to the GST antibody; after co-incubation of the recombinant proteins GmNPR1-GST and GmMYB306-His, a positive band of the recombinant protein GmMYB306-His was detected in the pull-down lane, while after co-incubation of the recombinant protein GmMYB306-His and GST protein, no positive band of the recombinant protein GmMYB306-His was detected in the pull-down lane, indicating that GmNPR1 and GmMYB306 can interact in vitro.

[0101] Example 5: Identification of downstream target genes of soybean GmMYB306 gene

[0102] The overexpression recombinant plasmid GmMYB306-Myc + recombinant plasmid GmPR1-LUC and the recombinant plasmid GmPR1-LUC + LUC empty effector vector were co-transformed into Agrobacterium GV3101 strain, and then co-transfected into Nicotiana benthamiana for transient expression. After culturing in the dark for 3 days, 1 mmol / LD-Luciferin solution was sprayed and imaging was performed using the Tianneng 5200 chemiluminescence system. The results are as follows: Figures 9-10 shown.

[0103] The results showed that compared with the leaves of Nicotiana benthamiana co-transfected with the recombinant plasmid GmPR1-LUC and the LUC empty effector vector, the chemiluminescence signal was significantly activated in the leaves co-transfected with the overexpressing recombinant plasmid GmMYB306-Myc and the recombinant plasmid GmPR1-LUC, indicating that GmMYB306 can regulate the expression of GmPR1.

[0104] Example 6: Overexpression of soybean GmMYB306 gene to resist Phytophthora sojae

[0105] (1) Culture medium configuration:

[0106] Growth medium preparation: 20 g sucrose, 3.21 g B5 salts, 0.6 g MES, and 8 g agar were diluted to 1 L of water, adjusted to pH 5.8, and sterilized. After cooling to room temperature, 6-BA was added to a final concentration of 1.67 g / L to obtain growth medium.

[0107] Preparation of root induction medium: 30g sucrose, 2.215g 1 / 2MS salt, 0.6g MES, 8g agar were diluted to 1L water, pH was adjusted to 5.8 and sterilized, and 1000mg cefoperazone and 500mg ampicillin sodium (C17 H 16 N2Na2O6S) to obtain root induction medium.

[0108] (2) Preparation of bacterial suspension: LUC empty effector vector, overexpression recombinant plasmid GmMYB306-Myc, and recombinant plasmid GmMYB306-RNAi were transformed into Agrobacterium K599 competent cells, and propagated to the bacterial suspension OD 600nm =0.8, and after collecting the bacteria by centrifugation, 2% MgCl2 solution (containing 0.04 g / L acetylsalicylic acid) was added for resuspending. The amount added was 10 mL of 2% MgCl2 solution per 1 g of bacteria to obtain LUC bacterial suspension, GmMYB306-Myc bacterial suspension, and GmMYB306-RNAi bacterial suspension, respectively.

[0109] (3) Genetic transformation: Healthy, plump, and disease-free soybean seeds of the Dongnong 50 variety were selected and sterilized with chlorine for 16 hours. They were then placed in a growth medium and naturally cultivated for 7 days to obtain soybean seedlings. When the soybean seedlings developed to a state where the bean body was bright green and the seed coat was tightly covered, the embryos of the soybean seedlings were removed from the cotyledons, and a 30 mm × 30 mm inoculation wound was created on the back of the cotyledons. 10 μL each of the LUC bacterial suspension, the GmMYB306-Myc bacterial suspension, and the GmMYB306-RNAi bacterial suspension were taken and accurately inoculated into the inoculation wound on the back of the cotyledons. After sealing, the suspensions were placed in a root induction medium and cultured for 21 days at a temperature of 25°C, a relative humidity of 70%, and a light intensity of 16 h / d. The corresponding soybean plants of the EV strain, the GmMYB306-OE strain, and the GmMYB306-RNAi strain were obtained.

[0110] (4) Qualitative determination of genetic transformation: The hairy roots of soybean plants of Dongnong 50, EV and GmMYB306-OE were collected and irradiated with GFP green fluorescence excitation light with an emission wavelength of 488 nm to observe the fluorescence of GFP protein in the hairy roots of soybean plants of each strain. At the same time, the hairy roots of soybean plants of Dongnong 50, EV and GmMYB306-RNAi were collected and the exogenous bar gene was detected in the hairy roots of soybean plants of each strain using bar protein rapid detection paper strips and agarose gel electrophoresis. The results are as follows: Figures 11-12 shown.

[0111] (5) Expression level detection: qRT-PCR was used to detect the expression level of GmMYB306 in the EV strain, GmMYB306-OE strain, and GmMYB306-RNAi strain. The nucleotide sequences of the qRT-PCR primers are shown in Table 3. The detection results are shown in Figure 13 shown.

[0112] (6) Resistance identification: The hairy roots of soybean plants of EV, GmMYB306-OE, and GmMYB306-RNAi were inoculated with Phytophthora sojae. 48 h after inoculation, the hairy roots of soybean plants of each strain were examined under a microscope for symptoms of disease. qRT-PCR was used to detect the expression levels of the internal reference genes GmTEF1 and GmPSPEL1 of Phytophthora sojae in soybean plants of each strain. The nucleotide sequences of the qRT-PCR primers are shown in Table 3. The results are shown in Table 3. Figures 14-15 shown.

[0113] Table 3 qRT-PCR primer sequences

[0114] name Sequence (5'~3') SEQ ID NO. GmMYB306-QF TGGCCAAGAAAGCCTTGTGT 25 GmMYB306-QR GAAGAGCCATGGTGTTTGGAG 26 GmTEF1-QF TGATCGTGCTGAACCACCC 27 GmTEF1-QR CGAGCGACGGTCCATCTT 28 GmPSEL1-QF CCGCGTACGTGGCTTTGGTGAG 29 GmPSEL1-QR ATCTTGGCGACTGAGGCTGCTTAC 30

[0115] The results of qualitative genetic transformation and expression level detection showed that the soybean plants of GmMYB306-OE and GmMYB306-RNAi lines were successfully genetically transformed;

[0116] Resistance identification results showed that 48 hours after inoculation, compared with the EV strain, the hairy roots of soybean plants in the GmMYB306-OE strain showed no obvious disease symptoms, while the hairy roots of soybean plants in the GmMYB306-RNAi strain showed severe disease and a dark brown surrounding area. The expression levels of the P. sojae internal reference genes GmTEF1 and GmPSPEL1, which can represent the accumulation of P. sojae biomass, showed that the biomass accumulation of P. sojae in the hairy roots of soybean plants in the GmMYB306-OE strain was significantly lower than that in the EV strain (**P < 0.01), while the biomass accumulation of P. sojae in the hairy roots of soybean plants in the GmMYB306-RNAi strain was significantly higher than that in the EV strain (**P < 0.01). This suggests that GmMYB306 is a positive regulator of the response to P. sojae infection.

[0117] As can be seen from the above examples, the present invention provides the use of overexpressing the soybean GmMYB306 gene in the preparation of products that enhance crop resistance to Phytophthora root rot. GmMYB306 can interact with the disease resistance-associated protein GmNPR1 and enhance soybean plant defense against Phytophthora sojae by regulating the expression of the crop disease progression-associated gene GmPR1, thereby playing an important role in enhancing soybean plant resistance to Phytophthora root rot.

[0118] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of overexpressed soybean GmMYB306 gene in the preparation of a product for improving crop resistance to soybean Phytophthora root rot, characterized in that: The nucleotide sequence of the soybean GmMYB306 gene is shown in SEQ ID NO.

1.

2. Use of the overexpressed soybean GmMYB306 gene in preparing a product for increasing the expression of crop disease-related genes, characterized in that: The nucleotide sequence of the soybean GmMYB306 gene is shown in SEQ ID NO.1; The crop disease course-related gene is the GmPR1 gene.

3. An overexpression recombinant plasmid, characterized in that: The overexpression recombinant plasmid includes the soybean GmMYB306 gene and an overexpression empty vector; The nucleotide sequence of the soybean GmMYB306 gene is shown in SEQ ID NO.1; The overexpression empty vector is pCAMBIA3301 vector.

4. The method for constructing the overexpression recombinant plasmid according to claim 3, characterized in that: The soybean GmMYB306 gene was connected to the empty overexpression vector to obtain an overexpression recombinant plasmid.

5. A primer pair, characterized in that: Includes upstream primers and downstream primers; The nucleotide sequence of the upstream primer is shown in SEQ ID NO.25; The nucleotide sequence of the downstream primer is shown in SEQ ID NO.

26.

6. Use of the primer pair according to claim 5 in detecting the expression level of the soybean GmMYB306 gene.

7. A recombinant bacterium, characterized in that The recombinant bacteria include the overexpression recombinant plasmid according to claim 3 and empty bacteria; The empty-loaded bacteria are Escherichia coli Trans1-T1 strains.

8. Use of the overexpression recombinant plasmid according to claim 3 or the recombinant bacterium according to claim 7 in improving crop resistance to soybean Phytophthora root rot.

9. Use of the overexpression recombinant plasmid according to claim 3 or the recombinant bacteria according to claim 7 for increasing the expression level of genes related to crop disease progression.

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