Application of soybean GmWRKY40 gene in soybean breeding

Abstract

The invention provides application of a soybean GmWRKY40 gene in soybean breeding, and belongs to the technical field of functional gene breeding. The nucleotide sequence of the soybean GmWRKY40 gene provided by the invention is as shown in SEQ ID NO: 1. According to the invention, the GmWRKY40 gene is constructed into a skeleton expression vector to obtain a recombinant vector, and the recombinant vector is transferred into soybean under the mediation of agrobacterium tumefaciens to obtain a transgenic plant with stable overexpression of the GmWRKY40 gene. Detection results show that overexpression of the GmWRKY40 gene can significantly improve the grain isoflavone content and the protein oil content of the transgenic soybean, improve the soybean quality and increase the soybean grain yield at the same time. Therefore, the soybean GmWRKY40 gene can be used for developing new varieties of high-quality soybeans.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional gene breeding, and specifically relates to soybean GmWRKY40 Application of genes in soybean breeding. Background Art

[0002] As a natural phytoestrogens, soy isoflavones play a positive role in human health. Isoflavones belong to flavonoid compounds and are one of the important secondary metabolites of the phenylalanine metabolic pathway. As a precursor of phytoalexins, they play an important role in resisting external biological and abiotic stresses. Soybeans are rich in isoflavones. Although isoflavones can also be synthesized in other plants, soybeans are still the only effective source of isoflavones for humans due to the imperfect isoflavone extraction process and low yield. The synthesis of soybean isoflavones is regulated by multiple genes, including transcription factors such as MYB, ZFP and WRKY. They regulate the production of isoflavones by binding to downstream target genes, thereby improving soybean quality and disease resistance. However, there are currently no reports on functional genes that regulate the biosynthesis of soybean isoflavones. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a soybean GmWRKY40 The invention discloses a gene which positively regulates the isoflavone content in soybean seeds, and provides a new means for cultivating high-quality gene resources and new soybean varieties.

[0004] The invention provides a soybean GmWRKY40 Gene, the soybean GmWRKY40 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0005] The present invention provides the soybean GmWRKY40 The protein encoded by the gene has an amino acid sequence as shown in SEQ ID NO: 2.

[0006] The invention provides a recombinant vector for expressing the protein.

[0007] Preferably, the soybean GmWRKY40 Gene.

[0008] The present invention provides a recombinant strain comprising the soybean GmWRKY40 Gene or the recombinant vector.

[0009] The present invention provides the soybean GmWRKY40 Application of the gene, the protein, the recombinant vector or the recombinant strain in soybean breeding.

[0010] Preferably, the soybean breeding includes increasing the isoflavone content in soybean seeds, increasing the protein content in soybean seeds and increasing soybean yield.

[0011] Preferably, the isoflavones include at least one of the following: daidzein, glycitein and genistein.

[0012] The present invention provides the soybean GmWRKY40 Application of the gene, the protein, the recombinant vector or the recombinant strain in constructing transgenic soybeans rich in isoflavones.

[0013] The present invention provides a method for increasing the content of soybean isoflavones, comprising the following steps: Soybean GmWRKY40 Gene construction recombinant expression vector; The constructed recombinant expression vector is transferred into soybean to obtain transgenic soybean plants; The transgenic soybean plants are grown and managed until maturity, and soybean seeds are harvested to obtain soybean seeds rich in soybean isoflavones.

[0014] The invention provides a soybean GmWRKY40 gene, the nucleotide sequence of which is shown in SEQ ID NO: 1. GmWRKY40 The gene was constructed into an overexpression recombinant vector and then introduced into soybean plants to obtain overexpression soybeans. GmWRKY40 The results of testing soybean isoflavone content in soybean plants overexpressing GmWRKY40 The gene significantly increased the content of different types of isoflavones and total isoflavones in the seeds of soybean plants compared with wild-type soybean plants. GmWRKY40 The contents of isoflavone components in the genetically modified soybean seeds are daidzein (DZ): 1043.94~1056.32μg / g, glycitein (GC): 679.85~976.53μg / g, genistein (GT): 1198.49~1705.47μg / g, and total isoflavone (TI): 3231.34~3429.26μg / g, which are 34.59%, 13.14%, 26.2% and 25.15% higher than those in wild-type soybeans, respectively. GmWRKY40 Genes can increase the content of soybean isoflavones and total isoflavones through positive regulation. GmWRKY40 The gene also increases soybean plant height and seed yield (including single plant seed weight and 100-seed weight) and increases protein accumulation in soybean seeds. GmWRKY40The gene can not only increase the isoflavone content in the seeds, but also improve the quality of soybeans and can be used to develop new high-quality soybean varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 for GmWRKY40 Excellent haplotype analysis results, where a: GmWRKY40 Gene structure; b: rs10262522 haplotype analysis results; **: significant at 0.01 level; Figure 2 Total RNA extraction from soybean (A) and soybean GmWRKY40 PCR amplification results of the gene (B); Figure 3 The PCR identification results of the transformed Agrobacterium EHA105, where A is the result of single enzyme digestion, 1 is the empty vector plasmid, and 2 is the plasmid after enzyme digestion; B is the result of PCR identification of the bacterial solution, M is the DNA marker; 1 is the positive control, 2 is the empty vector control (negative control); 3 is the water control (negative control); 4-13 are the PCR products of the bacterial solution containing the recombinant plasmid; Figure 4 To overexpress soybean GmWRKY40 PCR detection results of plants with Bar gene, where A is the PCR identification of Bar gene primers; B is GmWRKY40 PCR identification of gene-specific primers; M is DNA marker; 1 is the PCR product with positive plasmid as template (positive control), 2 is the PCR product with deionized water as template (negative control); 3-6 are overexpressed soybean GmWRKY40 PCR products using plant DNA of the gene as template; Figure 5 To overexpress soybean GmWRKY40 Results of fluorescence quantitative PCR detection of plants expressing the gene; CK1 is the PCR product using cDNA of wild-type plant leaves as template; CK2 is the PCR product using deionized water as template (negative control); 1-4 are PCR products using cDNA of overexpressed soybean GmWRKY40 PCR products using cDNA from leaves of genetic plants as templates; Figure 6 To overexpress soybean GmWRKY40 The results of semi-quantitative RT-PCR detection of genes in plants; CK1 is the PCR product with cDNA of wild-type plant leaves as template; CK2 is the PCR product with deionized water as template (negative control); 1-4 are the PCR products with cDNA of overexpressed soybean GmWRKY40 PCR products using cDNA from leaves of genetic plants as templates; Figure 7 To overexpress soybean GmWRKY40The results of plant detection of genes; A is the detection result of BAR protein by PAT / BAR gold label rapid immune detection kit, and B is the detection result of soybean overexpression by Western-Blot technology. GmWRKY40 The results of recombinant protein expression in plants expressing the gene; Figure 8 To overexpress soybean GmWRKY40 The results of isoflavone content determination in plants with the gene; Figure 9 To overexpress soybean GmWRKY40 Genetic plant growth diagram. DETAILED DESCRIPTION

[0016] The invention provides a soybean GmWRKY40The gene has a nucleotide sequence as shown in SEQ ID NO:1 (ATGGTCGTTCAACTCTTCAACACGGCAATCATAATGGATGAAAAGGTGGAAACTCTTAAAGCTGAGTTACAACGAGTGAGAGAGGAGAACAACACTCTAAGACTGATGCTTGAAGTTCTAAACAGCAAGTGCACAAAGCTTGAGATCCATCTTCAAGAGATAAACAAGGCACAACACAAGGGCATGAGTTCAAATCAAATAGGGTCAGTAACAGTACCACCTATGTTTGATACAAACAAGAGACCGAGACTAGAGCTTCCCACAGCAAAAAAGCCATTACAAATCTTTGTCAGAACACACCCCAAGGATGATAGTTTGATAGTAAAAGATGGCTATCAGTGGAGGAAGTATGGGCAGAAGGTTACCAAAGACAATGCTTCACCAAGAGCTTATTTCAGGTGCTCCATGGCTCCCATGTGCCCAGTCAAAAAGAAGGTGCAAAGATGCTTACATGATAAGTCAATCGTTGTTGCAACTTATGATGGAGAGCACAACCATGCGGCCATTCATGAGTCATCTTCATCCACATCTAAAGGCTCATCACCAGTGGTTAATAACTTACCTCTGATGACAAGCATATTAAATGACAAAGAACCCATGAACATTGGCTTGGCTCTTTCAGGGTGGTCTCAAACAGATCATAGGAGACATTGTGAAGATGCCATGCAACAGAATAATAATAATGGTGGTAGCAACATCAGAATTGAAGAATATGTAAGTTCTCTAATAAAAAATCCTGACTTCTCCATGTCATTAGCTGAAGCAGTTGTCCGCACCATCACTGGCCAGCAAAAGCAACAAGATATAAACCTTAATTTGGATCTTCCTGAAGAGTGA).

[0017] The present invention provides the said soybean GmWRKY40 The protein encoded by the gene, and the amino acid sequence of the said protein is as shown in SEQ ID NO:2 (MVVQLFNTAIIMDEKVETLKAELQRVREENNTLRLMLEV LNSKCTKLEIHLQEINKAQHKGMSSNQIGSVTVPPMFDTNKRPRLELPTAKKPLQIFVRTHPKDDSLIVKDGYQWRKYGQKVTKDNASPRAYFRCSMAPMCPVKKKVQRCLHDKSIVVATYD GEHNHAAIHESSSSTSKGSSPVVNNLPLMTSILNDKEPMNIGLALSGWSQTDHRRHCEDAMQQNNNNGGSNIRIEEYVSSLIKNPDFSMSLAEAVVRTITGQQKQQDINLNLDLPEE*).

[0018] The present invention provides a recombinant vector for expressing the protein described in the above technical solution.

[0019] In the present invention, the recombinant vector preferably comprises the soybean GmWRKY40 The present invention has no special restrictions on the type of backbone vector of the recombinant vector, and the corresponding recombinant expression vector can be flexibly selected according to the type of host cell. In the embodiment of the present invention, pCambia3300 plasmid is used as the backbone vector to illustrate soybean GmWRKY40 The biological function of the gene. The soybean GmWRKY40 The cloning site of the gene is H ind Ⅲ The present invention has no particular restrictions on the preparation method of the recombinant vector, and the recombinant vector construction method known in the art can be used, for example, using primers with enzyme cleavage sites to amplify soybean GmWRKY40 Gene fragment or gene synthesis method to obtain soybean with restriction enzyme cutting site GmWRKY40 The gene fragment is then subjected to restriction enzyme digestion with the same endonuclease as the backbone vector, and the resulting linear vector and gene fragment are connected. The connection product is verified and the soybean clone is successfully obtained. GmWRKY40 The recombinant vector of the gene fragment. GmWRKY40 Gene fragments can be codon-optimized according to the codon preference of the host cell to improve soybean GmWRKY40 The expression level of gene fragments.

[0020] The present invention provides a recombinant strain comprising the soybean GmWRKY40 Gene or the recombinant vector.

[0021] In the present invention, the host strain of the recombinant strain includes Agrobacterium strain EHA105 The present invention has no particular limitation on the construction method of the recombinant strain, and the recombinant strain can be transformed by a method known in the art, such as electroporation of Agrobacterium or heat shock method.GmWRKY40 The gene was transferred into the host plant, and then through multiplication and generation, soybean was successfully GmWRKY40 The gene is transferred into the host soybean genome to achieve soybean GmWRKY40 The gene is stably inherited in the host plant.

[0022] Given that soybean GmWRKY40 The gene has the function of positively regulating the isoflavone content in soybean seeds. The present invention provides a method for increasing the isoflavone content in soybeans, comprising the following steps: Soybean GmWRKY40 Gene construction recombinant expression vector; The constructed recombinant expression vector is transferred into soybean to obtain transgenic soybean plants; The transgenic soybean plants are grown and managed until maturity, and soybean seeds are harvested to obtain soybean seeds rich in soybean isoflavones.

[0023] In the present invention, the method for constructing a recombinant expression vector is the same as above and will not be described in detail here.

[0024] In the present invention, the method of transferring the constructed recombinant expression vector into soybean is preferably achieved by Agrobacterium-mediated transformation, specifically, the Agrobacterium strain carrying the recombinant expression vector is infected with soybean germination seeds, co-cultured to obtain transformed seedlings, screened in a screening medium containing glufosinate (PPT) (125 mg / L) for 7 to 10 days, and after elongation and rooting culture and seedling hardening, molecular detection is performed to obtain transgenic soybean plants. The molecular detection primers include 3300-WRKY40-486-F (5'-AGCAAGTGCACAAAGCTTGA-3', SEQ ID NO: 5) and 3300-WRKY40-486-R (5'-GCCAATGTTCATGGGTTCTT-3', SEQ ID NO: 6). The transgenic soybean plants were propagated and multiplied in a greenhouse to obtain T3 transgenic plants. PCR detection was performed on the T3 transgenic strains using primers 3300-WRKY40-486 and 3300-Bar-nos402, and the expected bands were detected, indicating that the GmWRKY40 gene had been successfully integrated into the soybean genome and could be inherited by offspring.

[0025] In the present invention, the soybean isoflavone content of the soybean seeds obtained from the T3 transgenic strain obtained above was determined by HPLC. The results showed that GmWRKY40The contents of isoflavone components in the transgenic soybean seeds of the gene are daidzein: 1043.94~1056.32μg / g, glycitein: 679.85~976.53μg / g, genistein: 1198.49~1705.47μg / g, total isoflavones: 3231.34~3429.26μg / g, which are significantly increased compared with the wild-type soybeans, with DZ, GC, GT and TI increased by 34.59%, 13.14%, 26.2% and 25.15%, respectively.

[0026] The present invention provides the soybean GmWRKY40 Application of the gene, the protein, the recombinant vector or the recombinant strain in constructing transgenic soybeans rich in isoflavones.

[0027] In the present invention, the method for constructing the isoflavone-rich transgenic soybean is the same as the above technical solution, which will not be described in detail here.

[0028] The present invention provides the soybean GmWRKY40 Application of the gene, the protein, the recombinant vector or the recombinant strain in soybean breeding.

[0029] In the present invention, the target soybean variety of soybean breeding preferably includes at least one of the following: rich in isoflavones, yield advantage and advantage in the proportion of protein and oil in soybean seeds. The isoflavones preferably include at least one of the following: daidzein, glycitein and genistein. The rich isoflavones means that compared with wild-type soybean plants, the isoflavone content in the seeds of transgenic soybean plants is significantly increased, daidzein is 1043.94~1056.32μg / g, glycitein is 679.85~976.53μg / g, genistein is 1198.49~1705.47μg / g, and total isoflavones is 3231.34~3429.26μg / g, which is significantly improved compared with wild-type soybeans, among which DZ, GC, GT and TI increased by 34.59%, 13.14%, 26.2% and 25.15% respectively.

[0030] In the present invention, the yield advantage refers to the increase in soybean GmWRKY40 The expression level of the gene in soybean significantly increased the grain weight per plant and the weight of 100 grains compared with soybeans before overexpression. In the present embodiment, biological yield-related indicators of transgenic soybean plants were measured respectively. The results showed that GmWRKY40 There was no significant difference in the number of pods and single grains between the transgenic soybean plants with the gene and the wild-type Williams 82, but the single-plant grain weight (17.1g) and 100-grain weight (18.8g) were significantly higher than those of the wild-type Williams 82.

[0031] In the present invention, the protein and oil content ratio advantage in soybean seeds refers to that the protein content ratio of transgenic soybean seeds is significantly increased compared with that of wild-type soybean seeds, and the oil content ratio also has an increasing trend.

[0032] In the present invention, the target soybean variety of soybean breeding also includes changes in plant type and flower development time. The plant type changes include increases in plant height and main stem node number of transgenic soybean plants. The changes in flower development time include delayed flower development time.

[0033] Soybean provided by the present invention GmWRKY40 By stably overexpressing the gene in soybean plants, it is beneficial to greatly increase the isoflavone content in the seeds and increase the protein and oil content in the soybeans, thereby improving the soybean variety and also increasing the soybean yield. The present invention can be used for the development of new high-quality soybean varieties.

[0034] The soybean provided by the present invention is described below in conjunction with the embodiments GmWRKY40 The application of the gene in soybean breeding is described in detail, but they should not be understood as limiting the scope of protection of the present invention.

[0035] The agarose and nucleic acid dye used in the present invention are provided by Rambolide Company; The marker used—LD DSTM 2000—was purchased from Guangzhou Dongsheng Company; The RNA extraction reagent RNAiso Plus used was purchased from TaKaRa; Restriction enzymes were provided by Thermo Fisher Scientific; The KOD OneTM PCR Master Mix -Blue-enzyme used for high-fidelity PCR amplification, the common reverse transcription kit ReverTra Ace qPCR RT Master Mix with gDNA Remover, and the reagent THUNDERBIRD® Next SYBR® qPCR Mix used for fluorescence quantification were all purchased from TOYOBO. The homologous recombination ligation kit ClonExpress II One Step Cloning Kit and the 2 × Rapid Taq Master Mix used for bacterial liquid rapid PCR were purchased from Nanjing Novozyme Biotechnology Co., Ltd. Gel recovery and plasmid extraction kits were provided by OMEGA; The reagents used for soybean genetic transformation were purchased from Sigma; The synthesis of gene primers and gene sequencing were completed by Beijing Ruibo Company.

[0036] Agilent 1100 high performance liquid chromatography (HPLC) detection platform was used to quantitatively analyze the different components of soybean isoflavones in the test materials.

[0037] Isoflavone HPLC analysis standards were provided by Shanghai Xinyu Biotechnology Co., Ltd. The near-infrared grain analyzer used in the experiment was provided by Beijing FOSS Company.

[0038] Unless otherwise specified, other experimental reagents and instruments can be purchased through commercial channels.

[0039] The soybean varieties Williams 82, Zhongdou 27, and Jiunong 20 are soybean varieties well known in the art and can be purchased through commercial channels.

[0040] RNA was extracted using TaKaRa RNAiso Plus, and reverse transcribed into cDNA using a common reverse transcription kit, ReverTra AceqPCR RT Master Mix with gDNA Remover, for cloning soybean GmWRKY40 Gene, primers synthesized by Beijing Ruibo Company were used to clone the target gene fragment through KOD OneTM PCR Master Mix -Blue-high-fidelity enzyme; By restriction endonuclease Hind Ⅲ The pCambia3300 vector was digested with a single enzyme.

[0041] Will GmWRKY40 The gene was constructed into an expression vector and transformed into DH5α Escherichia coli competent cells. The plasmid was extracted from the positive strain and the recombinant plasmid was transferred into EHA105 competent cells. Subsequently, it was introduced into Williams 82 soybean through Agrobacterium-mediated semi-seed transformation to obtain stable transgenic plants.

[0042] Agilent 1100 HPLC detection platform was used to quantitatively analyze the different components of soybean isoflavones in the test materials.

[0043] Overexpression GmWRKY40 The gene can significantly increase the isoflavone content in seeds and improve the quality of soybeans. The invention can be used for the development of new high-quality soybean varieties.

[0044] Example 1 GmWRKY40 Acquisition of genes Using high-isoflavone soybean Zhongdou 27 as the recipient parent and low-isoflavone variety Jiunong 20 as the recurrent parent, a BC2F5 near-isogenic line population containing 175 families was constructed through multi-generation backcrossing combined with marker screening. Genotyping was performed on each family in the population, and the ICIM-ADD method in the bip module built into IciMapping v4.2 was used to locate the QTL. It was found that qISO14-1 was related to isoflavone content, and a candidate gene was found in the 161 kb segment of the QTL. GmWRKY40 The sequence variation of Figure 1 ).

[0045] Example 2 change GmWRKY40 Construction of genetically modified soybean plants.

[0046] 1. GmWRKY40 Gene cloning 1) Total RNA extraction (Trizol method) ① Use scissors to cut the tender leaves of Zhongdou 27 and put them into a 2.0ml centrifuge tube. Quickly put them into a liquid nitrogen box for freezing and grinding. Add 1mL of pre-cooled Trisol extract to the centrifuge tube, shake well to mix, and place on ice for 5min. ② Add 200 μL of chloroform, shake vigorously to mix thoroughly, let stand for 3-5 minutes, and then centrifuge at 4°C and 12,000 rpm for 15 minutes; ③ Extract the supernatant and place it in a new centrifuge tube, add an equal volume of isopropanol, shake evenly for 2-3 minutes, and let it stand at -20℃ for 20-30 minutes; ④ Centrifuge at 4°C, 12,000 rpm for 10 min; ⑤ Discard the supernatant, add 1 mL of 75% ethanol (dilute the anhydrous ethanol with DEPC water), shake and centrifuge at 12,000 rpm at 4°C for 10 min; ⑥ Repeat step ⑤ once; ⑦ Discard the supernatant, leave it in the air for 2 minutes, remove the remaining supernatant with a pipette, and dry it on ice for 10 minutes; ⑧ After drying, add 20 μl DEPC water to dissolve and check the quality by gel electrophoresis ( Figure 2 Middle A).

[0047] 2) Total RNA reverse transcription Total RNA was reverse transcribed into cDNA according to the reaction procedure of TOYOBO's cDNA synthesis kit (ReverTra Ace qPCR RT Master Mix with gDNA Remover, CAS No.: FSQ-301). The specific steps are as follows: ① RNA denaturation: Place the RNA in a 65°C metal bath, keep warm for 5 min, and cool in ice.

[0048] ② Removal of genomic DNA: Use ReverTra Ace qPCR RT Master Mix with gDNARemover kit to remove the remaining genomic DNA. The reaction system is shown in Table 1: Table 1 Reaction system and procedure for genomic DNA removal

[0049] ③ Reverse transcription reaction: Prepare the reverse transcription reaction system as shown in Table 2. After the reaction is completed, store it at -20°C for future use.

[0050] Table 2 Reverse transcription reaction system and procedure

[0051] Select the master copy gene sequence and design a pair of GmWRKY40 Primers were placed at the 5' and 3' ends of the CDS region and appropriate restriction sites were introduced. GmWRKY40 Primers for gene cloning: GmWRKY40 -F:TCGAGCTCCGTCGACAAGCTTATGGTCGTTCAACT(SE ID NO:3); GmWRKY40 -R: GCCCTTGCTCACCATAAGCTTTCACTCTTCAGGAA (SE ID NO:4); The cloning PCR reaction system refers to the kit instructions. The PCR reaction conditions are: 98℃ pre-denaturation for 3min, 98℃ denaturation for 10s, 58℃ annealing for 5s, 68℃ extension for 5s, amplification for appropriate times; 68℃ final extension for 10min, and the full-length CDS sequence of the gene was obtained (837bp) Figure 2 In B), the target gene was recovered and sequenced, and the sequencing result (SEQ ID NO: 1) was consistent with Phytozme The target sequence was compared with the reference sequence in the database and the result was completely consistent with the reference sequence. Store at 4℃.

[0052] 2. GmWRKY40 Gene vector construction Utilization of circular pCambia3300 plasmid hind Ⅲ The enzyme was used for single enzyme digestion, and the digestion was carried out at 37℃ for 2~4h and then separated by gel electrophoresis ( Figure 3), the recovered target fragment was connected with the linearized pCambia3300 vector fragment by homologous recombination to obtain a connection product. The connection system is shown in Table 3 with reference to the relevant instructions.

[0053] Table 3 Homologous recombination ligation reaction system and procedure

[0054] The ligation product was transformed into DH5α competent cells, and the positive strain was identified and sequenced correctly, indicating that pCambia3300 was successfully constructed. -GmWRKY40 The recombinant plasmid was then extracted and transformed into Agrobacterium EHA105. The Agrobacterium transformation method is as follows: ① Take the competent Agrobacterium stored at -80℃ and place it at room temperature or in the palm of your hand for a while until it partially melts. When it is in a mixture of ice and water, insert it into ice.

[0055] ② Add 0.01-1 μg plasmid DNA per 100 μL competent medium, stir the bottom of the tube by hand to mix, and place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes.

[0056] ③ Add 700 μL of LB liquid culture medium without antibiotics and culture at 28°C with shaking for 2 to 3 hours.

[0057] ④ Centrifuge at 6000 rpm for one minute to collect the bacteria, retain about 100 μL of the supernatant, gently blow to resuspend the bacteria, and spread it on the LB plate containing kanamycin and rifampicin antibiotics. Place it upside down in a 28℃ incubator for 2-3 days to observe the growth of bacterial plaques.

[0058] ⑤ After the colonies grow, pick a single plaque in the clean bench and inoculate it into 1 mL of LB liquid culture medium containing kanamycin (50 μg / mL) and rifampicin (20 μg / mL), shake and culture at 200 rpm at 28°C for 24-48 h, and perform bacterial liquid PCR verification.

[0059] ⑥ After successful PCR verification, bacteria are preserved.

[0060] The results showed that the recombinant plasmid pCambia3300- GmWRKY40 Successfully transformed into Agrobacterium tumefaciens EHA105 competent cells ( Figure 3 ).

[0061] 3. GmWRKY40 Genetic transformation 1) Preparation of bacterial culture A single colony of EHA105 carrying the target gene was selected and inoculated into LB liquid medium containing kanamycin (50 μg / mL) and rifampicin (20 μg / mL), and cultured overnight at 28°C and 200 rpm. Then the bacterial solution was transferred to a 100 ml breathable Erlenmeyer flask, 50 ml of LB liquid medium was added, and the mixture was shaken to A. 600 =0.6~0.8, place at 5000 rpm, centrifuge for 10 min, resuspend the bacterial solution in 100 mL CCM co-culture liquid medium and add 24.5 μL acetosyringone and 200 μL 6-BA for later use.

[0062] 2) Agrobacterium-mediated half-seed transformation method ① Seed sterilization Take mature Williams 82 soybean seeds with smooth, plump and spot-free appearance, put them in a culture dish, and place them in a desiccator in a fume hood. Open the lid of the culture dish halfway, measure 96 ml of NaClO and put it in a beaker, and place the beaker in the middle of the culture dish. When everything is ready, add 6 ml of concentrated hydrochloric acid (HCl) to the NaClO, quickly cover the desiccator, sterilize for 16 to 18 hours, take out, place in a clean bench and blow for 30 minutes to 1 hour to remove residual chlorine, and seal it for later use.

[0063] ② Seed germination and transformation receptor preparation Use tweezers to sow the sterilized seeds in the germination solid culture medium in the clean bench. When the seeds germinate to the point where the cotyledons are about to break through the seed coat, use tweezers, scalpels and other tools to remove the seed coat, retain the hypocotyl 3 mm away from the cotyledons, cut the cotyledons along the hypocotyl direction, use a scalpel to gently scrape off the apical buds and axillary buds between the cotyledons and the growth point, dip in the CCM resuspension containing Agrobacterium, and gently make 3 to 5 wounds at the cotyledon node. The remaining part is used as an explant for infection.

[0064] ③ Infection The explants and resuspension were placed in a sterile tissue culture bottle, sealed, and placed in a 28°C constant temperature shaker at 100 rpm for infection for 30 min.

[0065] ④ Co-cultivation The infected explants were washed once with sterile distilled water, the surface liquid was blotted with sterile filter paper, two layers of sterile filter paper were spread on the solid culture medium, the infected explants were placed with the cut side facing downward on a culture dish of CCM solid culture medium, and cultured in the dark in the tissue culture room for 3 days.

[0066] ⑤ Cultivation and screening of transformed seedlings After the co-cultivation, the cotyledon node explants were washed three times with sterile water, dried with sterile paper, and laid flat on the recovery medium with the cut surface facing downward for 7 days. The recovered cotyledon node explants were transferred to the screening medium containing glufosinate (PPT) (125 mg / L) for 7-10 days.

[0067] ⑥Elongation and rooting The selected explants with PPT resistance were transferred to elongation medium and subcultured every 7 days. When the buds elongated to 3-4 cm, they were cut off close to the base and transferred to rooting medium for rooting culture.

[0068] ⑦Seedling training When the seedlings are strong enough, they are transferred to vermiculite and cultured under weak light conditions for 3 days, and then transplanted to soil and cultured until maturity.

[0069] 4. GmWRKY40 Detection of genetically modified soybeans 1) PCR (polymerase chain reaction) analysis of transgenic plants DNA of transgenic and wild soybean Williams 82 was extracted according to the general method, and PCR identification was performed using DNA as a template, specific detection primers and universal Bar primers designed according to the gene and vector sequences. The reaction system is shown in Table 4: Table 4 PCR reaction system

[0070] change GmWRKY40 Plant identification primers: 3300- WRKY40 -486-F: 5'-AGCAAGTGCACAAAGCTTGA-3' (SEQ ID NO: 5); 3300- WRKY40 -486-R: 5'-GCCAATGTTCATGGGTTCTT-3' (SEQ ID NO: 6); 3300- Bar -nos402-F: 5'-GCGGTACCGGCAGGCTGAAG-3' (SEQ ID NO:7); 3300- Bar -nos402-R: 5'-CCGCAGGAACCGCAGGAGTG-3' (SEQ ID NO: 8); Reaction conditions: Bar Primer reaction program: 95℃ 3min; 35 cycles: 95℃ 15s, 54℃ 15s, 72℃ 15s; 72℃ 5min, 12℃ storage. 3300- WRKY40-486 primer reaction program: 95℃ 3min; 35 cycles: 95℃ 15s, 58℃ 15s, 72℃ 15s; 72℃ 5min, stored at 12℃. After the reaction, 6μL of PCR product was subjected to 2% agarose gel electrophoresis for detection.

[0071] After PCR and Bar test strip testing, 17 strains of T 0 The transgenic plants, T 0 The positive plants of the first generation were propagated in the greenhouse and then grown using 3300- WRKY40 -486 primers and 3300- Bar -nos402 primer pair T 3 PCR detection of transgenic lines ( Figure 4 The test results of some plants are shown in Figure 2. GmWRKY40 The gene has been successfully integrated into the soybean genome and can be inherited by offspring.

[0072] 2) Analysis of transcriptional levels of target genes in transgenic plants ① Transfer GmWRKY40 Fluorescence quantitative PCR of genetically modified soybean A. Total RNA was extracted and reverse transcribed into cDNA. The cDNA was diluted and adjusted to a concentration of 300 ng / μl as a template for fluorescence quantitative PCR.

[0073] B. Real-time fluorescence quantitative PCR Transfer GmWRKY40 The cDNA of leaf tissue of gene plants was used as template and real-time fluorescence quantitative PCR was used to detect GmWRKY40 The expression of genes and the internal reference gene Actin The average expression level of the target gene and the internal reference gene was taken as the reference (three replicates were set for each treatment).

[0074] GmWRKY40 Gene fluorescence quantitative primers: qGmWRKY40-F: 5'-TCCACATCTAAAGGCTCATCACCAG-3' (SEQ ID NO: 9); qGmWRKY40-R: 5'-GCATGGCATCTTCACAATGTCTCC-3' (SEQ ID NO: 10); Actin Gene fluorescence quantitative primers: Actin4-F: 5'-GTTTCAAGCTCTTGCTCGTAATCA-3' (SEQ ID NO: 11); Actin4-R: 5'-GTGTCAGCCATACTGTCCCCATTT-3' (SEQ ID NO: 12).

[0075] The reaction system is as follows: THUNDERBIRD ® Next SYBR ® qPCR Mix 10 μL; ddH 2 O 6.4μL; upstream primer (10μmol / L) 0.8μL; downstream primer (10μmol / L) 0.8μL; cDNA template 2μL. PCR program includes: 95℃ 30s; 95℃ 5s, 60℃ 30s, 72℃ 15s, 40 cycles. The CT value in the relative quantitative ΔΔCT method is the average of three experimental replicates. The relative copy number is calculated using the formula 2 -ΔΔCT (ΔΔCT=CT 目的基因 - CT 内参Actin基因 ).

[0076] The present invention uses 11 strains of T 3 The cDNA of the positive plants screened by PCR was used for RT-qPCR detection. The results showed that the transgenic soybean GmWRKY40 The expression level was generally higher than that of wild-type soybean, especially in transgenic plants. GmWRKY40 The expression level exceeded the wild-type plant by 10 times as the threshold for further screening, and finally GmWRKY40 Four transgenic plants with high gene expression were obtained, including GmWRKY40 The highest gene expression level was 53 times that of wild-type soybean ( Figure 5 ).

[0077] ② Transfer GmWRKY40 Semi-quantitative PCR detection of genetically modified soybean plants Take 4 strains of T 3 The young leaves of positive plants were used for transfer GmWRKY40 cDNA of leaf tissue of normal cultivated plants was used as template for semi-quantitative PCR amplification. The amplification results were verified by 2% agarose gel electrophoresis and the internal reference gene was used as template for semi-quantitative PCR amplification. Actin The average expression level of Figure 6 ). The primer sequence, system and reaction procedure are the same as those of the above-mentioned fluorescence quantitative PCR.

[0078] 3) Western-Blot detection of transgenic plants Protein extraction: Take 0.3-0.5g of fresh leaves and grind them with liquid nitrogen until they are crushed. Add 400ul of protein extraction solution, mix well, vortex, and ice bath for 10min, centrifuge at 13000 rpm at 4℃ for 15min, aspirate the supernatant into a 1.5ml protease-free centrifuge tube, take 80μl of the above supernatant, add 20μl of protein loading buffer, mix well, boil in water bath for 10min, centrifuge at 12000 rpm for 30s, and aspirate 20μl of supernatant for spotting; Prepare gel: Align a clean glass plate and place it on the gel rack. Pour the prepared separation gel into the gap of the glass plate to the edge of the green line. Quickly add distilled water along the upper edge. Let the gel stand for about 10 minutes. Pour out the upper layer of distilled water and dry it with filter paper. Add concentrated gel until it overflows, insert a comb, and let the gel last for about 15 minutes. Spotting: Add 1× running buffer to the electrophoresis tank, quickly pull out the comb vertically, and spot samples vertically against the wall; Gel running: Use 80V voltage for concentrated gel. After the sample runs into the separation gel, observe whether the marker runs away to adjust the voltage. Use 120V voltage for separation gel. Select the electrophoresis time according to the size of the protein. Take out the gel and transfer the membrane: carefully pry up the small glass plate, cut off the concentrated gel left on the large glass plate, prepare 2 WB special transfer sponge pads and 1 PVDF membrane, cut to the same size as the gel, place the PVDF membrane in methanol in advance, and place the sponge pad in the transfer solution to soak; The order of membrane transfer from negative electrode to positive electrode is: sponge, glue, PVDF membrane, sponge. Each layer needs to be driven away with a glass rod to remove bubbles. Use 400mA current to transfer the membrane, which takes about 30 minutes. Blocking: After transfer, take out the PVDF membrane and place it on a plate with blocking solution, and block it at 60 rpm for 1 hour at room temperature; Primary antibody reaction: Place the diluted primary antibody and PVDF membrane into a plate and shake at 60 rpm for 1 h at room temperature; Wash the membrane: Pour out the primary antibody in the dish, add 20 ml TBST again, shake at 60 rpm for 10 min at room temperature to wash the membrane, repeat the previous step, pour out the TBST and add 20 ml TBS to wash the membrane again for 10 min; Secondary antibody reaction: Place the diluted secondary antibody and PVDF membrane into a plate and shake at 60 rpm for 1 h at room temperature; Membrane washing: the same as the above membrane washing operation; development.

[0079] The gel electrophoresis of RT-PCR products showed that under the same conditions of internal reference amplification, no endogenous GmWRKY40 The expression of the gene was observed in both wild-type plants (CK2) and samples 1-4. GmWRKY40The expression of genes in transgenic plants was stronger than that in wild-type plants ( Figure 6 ), which is consistent with the results of RT-qPCR, indicating that GmWRKY40 The gene was successfully overexpressed.

[0080] The 11 strains that were positive by quantitative PCR were GmWRKY40 Genetically modified soybean plants were tested for BAR protein using the Agene PAT / BAR Gold Standard Immunoassay Rapid Detection Kit ( Figure 7 A), followed by Western-Blot detection of the tag gene, and bands were detected in 8 strains ( Figure 7 B in the figure shows some test results). This indicates that the marker gene successfully encodes protein products in the transgenic plants.

[0081] Example 2 GmWRKY40 Identification of isoflavone content in genetically modified soybeans 1) Soy isoflavone extraction Harvest T 3 Transfer GmWRKY40 Genetically modified soybean plant seeds, after drying soybean grains, select mature, plump and disease-free grains and grind them into powder. Accurately weigh 0.1g of sample in a 50mL centrifuge tube, add 7mL hydrochloric acid solution (2mol / L), vortex and shake for 10s to mix thoroughly, put it in a 90℃ water bath for acid leaching for 90min, then cool to room temperature, add about 3.5mL sodium hydroxide solution (4mol / L) until the pH is nearly neutral, then add 80% anhydrous ethanol to 25mL, invert and mix, and let stand for 30min for extraction. The extracted liquid was filtered with a 0.22μm syringe filter and injected into a 1.5mL liquid phase injection bottle and sealed. Store at 4℃ for testing, and repeat the test 3 times for each test material.

[0082] 2) Qualitative and quantitative analysis of soybean isoflavones Agilent 1100 high performance liquid chromatography (HPLC) detection platform was used to quantitatively analyze the different components of soybean isoflavones in the test materials. The detection conditions were as follows: chromatographic grade methanol and sterile deionized water were used as mobile phases. The liquid phase system was first flushed with chromatographic grade methanol. After the absorbance and pressure were stable, the ratio of methanol and sterile water was adjusted to a constant elution ratio of 1:1, the flow rate was 1mL / min, the column temperature was 35℃, the detection wavelength of the ultraviolet detector was 254nm, the injection volume was 10 μL, and the approximate peak time of each component of machine isoflavones was determined using isoflavone analysis standards, and the sample loading time was determined. The peak times of DZ, GC and GT were 7min30s, 8min and 11min, respectively, and the analysis time of a single sample was 15min.

[0083] The peak area method was used to calculate the content of each component of isoflavones. GmWRKY40 The contents of isoflavone components in the genetically modified soybean seeds were as follows: Daidzein (DZ): 1043.94 - 1056.32 μg / g, Glycitein (GC): 679.85~976.53 μg / g, Genistein (GT): 1198.49 ~1705.47 μg / g, Total Isoflavone (TI): 3231.34~3429.26 μg / g, which were significantly improved compared with the wild-type soybeans, with DZ, GC, GT and TI increasing by 34.59%, 13.14%, 26.2% and 25.15%, respectively ( Figure 8 ).

[0084] Example 3 GmWRKY40 Identification of agronomic traits of transgenic soybean Investigation transfer GmWRKY40 Gene T 3 After the plants matured, the transgenic positive plants were tested (mainly plant height, number of nodes, number of branches, number of pods, number of grains, and grain weight) to analyze their biological yield; the protein and oil content of transgenic and wild-type Williams 82 seeds was determined using a near-infrared grain analyzer.

[0085] It can be seen from Table 5 that GmWRKY40 Compared with the wild-type Williams 82, the transgenic GmWRKY40 The plant height and number of main stem nodes of transgenic soybean plants were significantly higher than those of wild type, while the number of branches was lower than that of wild type. Figure 9 It can be seen that the GmWRKY40There are differences in flower development between transgenic soybean plants and wild-type Williams 82. GmWRKY40 The transgenic soybean plants entered floral development significantly later than the wild type Williams 82.

[0086] Table 5 Significance analysis of the difference between transgenic and wild-type soybean plant height

[0087] Note: The data in the table are the average values ​​of 8 individual plants. # It indicates significant at the 0.05 level.

[0088] It can be seen from Table 6 that GmWRKY40 Compared with the wild-type Williams 82, the number of pods and single grains of the transgenic soybean plants had no significant difference, but the single-plant grain weight and 100-grain weight were significantly higher than those of the wild-type Williams 82.

[0089] Table 6 Significance analysis of differences in grain weight, grain number and pod number between transgenic and wild-type soybeans

[0090] Note: The data in the table are the average values ​​of 8 individual plants. # It indicates significant at the 0.05 level.

[0091] In order to compare the difference in soybean quality between transgenic and wild type, the transgenic GmWRKY40 Protein and oil content of genetically modified and wild-type Williams 82 seeds. It can be concluded from Table 7 that the protein content of genetically modified soybean seeds is significantly higher than that of wild-type seeds, and the oil content also tends to increase. The experimental results show that GmWRKY40 The expression of the gene can increase the accumulation of protein and oil in soybean seeds.

[0092] Table 7 Significance analysis of differences in seed quality between transgenic and wild-type soybeans

[0093] Note: The data in the table are the average values ​​of 6 individual plants. # It indicates significant at the 0.05 level.

[0094] Based on the results of the above embodiments, it can be seen that GmWRKY40 The gene can positively regulate the accumulation of soybean isoflavones and improve the quality of soybeans.

[0095] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A soybean GmWRKY40 A gene characterized by The soybean GmWRKY40 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The soybean according to claim 1 GmWRKY40 The protein encoded by the gene is characterized in that The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. A recombinant vector expressing the protein according to claim 2.

4. The recombinant vector according to claim 3, characterized in that: Comprising the soybean according to claim 1 GmWRKY40 Gene.

5. A recombinant strain, characterized in that: Comprising the soybean according to claim 1 GmWRKY40 Gene or the recombinant vector according to claim 3 or 4.

6. The soybean according to claim 1 GmWRKY40 Use of the gene, the protein according to claim 2, the recombinant vector according to claim 3 or 4 or the recombinant strain according to claim 5 in soybean breeding.

7. The use according to claim 6, characterized in that: The soybean breeding includes increasing the content of isoflavones in soybean grains, increasing the content of protein in soybean grains and increasing soybean yield.

8. The use according to claim 7, characterized in that: The isoflavones include at least one of the following: daidzein, glycitein and genistein.

9. The soybean according to claim 1 GmWRKY40 Use of the gene, the protein according to claim 2, the recombinant vector according to claim 3 or 4 or the recombinant strain according to claim 5 in constructing transgenic soybeans rich in isoflavones.

10. A method for increasing the content of soybean isoflavones, characterized in that: The following steps are involved: Soybean GmWRKY40 Gene construction recombinant expression vector; The constructed recombinant expression vector is transferred into soybean to obtain transgenic soybean plants; The transgenic soybean plants are grown and managed until maturity, and the soybean grains are harvested to obtain soybean grains rich in isoflavones.

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