Application of soybean GmWRKY40 gene in soybean breeding

Abstract

The present invention provides soybean GmWRKY40 The application of genes in soybean breeding belongs to the technical field of functional gene breeding. GmWRKY40 The nucleotide sequence of the gene is shown in SEQ ID NO: 1. GmWRKY40 The gene was constructed into a backbone expression vector to obtain a recombinant vector, which was then transferred into soybeans mediated by Agrobacterium tumefaciens to obtain stable overexpression. GmWRKY40 The test results showed that GmWRKY40 Overexpression of the gene can significantly increase the isoflavone content and protein and oil content of transgenic soybean seeds, improve soybean quality, and also increase soybean seed yield. GmWRKY40 The genes can be used to develop new high-quality soybean varieties.

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] Soy isoflavones, as natural phytoestrogens, have positive effects on human health. Isoflavones belong to the flavonoid family and are important secondary metabolites of the phenylalanine metabolic pathway. As precursors of phytoalexins, they play a crucial role in protecting against biotic and abiotic stresses. Soybeans are rich in isoflavones. Although other plants can also synthesize isoflavones, soybeans remain the only effective source of isoflavones for humans due to imperfect extraction processes and low yields. The synthesis of soy isoflavones is regulated by multiple genes, including transcription factors such as MYB, ZFP, and WRKY. These factors regulate isoflavone production by binding to downstream target genes, thereby improving soybean quality and disease resistance. However, no functional genes regulating soybean isoflavone biosynthesis have been reported. Summary of the Invention

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

[0004] The present 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:

[0014] Soybeans GmWRKY40 Gene construction recombinant expression vector;

[0015] The constructed recombinant expression vector is transferred into soybean to obtain transgenic soybean plants;

[0016] The transgenic soybean plants are grown and managed until maturity, and soybean seeds are harvested to obtain soybean seeds rich in soybean isoflavones.

[0017] The present invention provides a soybean GmWRKY40 The nucleotide sequence of the gene 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 the gene showed that GmWRKY40 The content of different types of isoflavones and total isoflavones in the seeds of soybean plants were significantly increased compared with wild-type soybean plants. GmWRKY40 The contents of isoflavone components in 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 isoflavones (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 Gene can increase the content of soybean isoflavones and total isoflavones through positive regulation. GmWRKY40 The gene also increases soybean plant height and grain yield (including single plant grain weight and 100-grain weight) and increases protein accumulation in soybean grains.GmWRKY40 The gene can not only increase the isoflavone content in seeds, but also improve the quality of soybeans, and can be used to develop new high-quality soybean varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 for GmWRKY40 Excellent haplotype analysis results, where a: GmWRKY40 Gene structure; b: rs10262522 haplotype analysis results; **: significant at the 0.01 level;

[0019] Figure 2 Total RNA extraction for two samples of soybean (A) and soybean GmWRKY40 PCR amplification results of the gene (B);

[0020] Figure 3 The PCR results of transformed Agrobacterium EHA105 are shown, where A is the result of single enzyme digestion, 1 is the empty plasmid, and 2 is the plasmid after enzyme digestion; B is the result of PCR identification of the bacterial solution, and M is a 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;

[0021] 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 to 6 are overexpressed soybean GmWRKY40 PCR products using plant DNA of the gene as template;

[0022] Figure 5 To overexpress soybean GmWRKY40 Results of fluorescence quantitative PCR detection of plants expressing the gene; CK1 is the PCR product using cDNA from wild-type plant leaves as template; CK2 is the PCR product using deionized water as template (negative control); 1-4 are the PCR products using cDNA from overexpressing soybean GmWRKY40 PCR products using cDNA from leaves of genetically modified plants as templates;

[0023] Figure 6 To overexpress soybean GmWRKY40 The results of semi-quantitative RT-PCR detection of genes in plants; CK1 is the PCR product with wild-type plant leaf cDNA as template; CK2 is the PCR product with deionized water as template (negative control); 1-4 are the PCR products with overexpression soybean GmWRKY40 PCR products using cDNA from leaves of genetically modified plants as templates;

[0024] Figure 7 To overexpress soybean GmWRKY40 The results of plant detection of genes; A is the detection result of BAR protein by PAT / BAR gold label rapid immune detection kit, B is the detection result of overexpressed soybean by Western-Blot technology GmWRKY40 Detection results of recombinant protein expression in plants expressing the gene;

[0025] Figure 8 To overexpress soybean GmWRKY40 The results of isoflavone content determination in plants with the gene;

[0026] Figure 9 To overexpress soybean GmWRKY40 Gene plant growth diagram. DETAILED DESCRIPTION

[0027] The present invention provides a soybean [[ID=4l]]GmWRKY40The gene has a nucleotide sequence as shown in SEQ ID NO:1 (ATGGTCGTTCAACTCTTCAACACGGCAATCATAATGGATGAAAAGGTGGAAACTCTTAAAGCTGAGTTACAACGAGTGAGAGAGGAGAACAACACTCTAAGACTGATGCTTGAAGTTCTAAACAGCAAGTGCACAAAGCTTGAGATCCATCTTCAAGAGATAAACAAGGCACAACACAAGGGCATGAGTTCAAATCAAATAGGGTCAGTAACAGTACCACCTATGTTTGATACAAACAAGAGACCGAGACTAGAGCTTCCCACAGCAAAAAAGCCATTACAAATCTTTGTCAGAACACACCCCAAGGATGATAGTTTGATAGTAAAAGATGGCTATCAGTGGAGGAAGTATGGGCAGAAGGTTACCAAAGACAATGCTTCACCAAGAGCTTATTTCAGGTGCTCCATGGCTCCCATGTGCCCAGTCAAAAAGAAGGTGCAAAGATGCTTACATGATAAGTCAATCGTTGTTGCAACTTATGATGGAGAGCACAACCATGCGGCCATTCATGAGTCATCTTCATCCACATCTAAAGGCTCATCACCAGTGGTTAATAACTTACCTCTGATGACAAGCATATTAAATGACAAAGAACCCATGAACATTGGCTTGGCTCTTTCAGGGTGGTCTCAAACAGATCATAGGAGACATTGTGAAGATGCCATGCAACAGAATAATAATAATGGTGGTAGCAACATCAGAATTGAAGAATATGTAAGTTCTCTAATAAAAAATCCTGACTTCTCCATGTCATTAGCTGAAGCAGTTGTCCGCACCATCACTGGCCAGCAAAAGCAACAAGATATAAACCTTAATTTGGATCTTCCTGAAGAGTGA).

[0028] The present invention provides the soybean GmWRKY40 protein encoded by the gene, and the amino acid sequence of the protein is as shown in SEQ ID NO:2 (MVVQLFNTAIIMDEKVETLKAELQRVREENNTLRLMLEV

[0029] LNSKCTKLEIHLQEINKAQHKGMSSNQIGSVTVPPMFDTNKRPRLELPTAKKPLQIFVRTHPKDDSLIVKDGYQWRKYGQKVTKDNASPRAYFRCSMAPMCPVKKKVQRCLHDKSIVVATYD GEHNHAAIHESSSSTSKGSSPVVNNLPLMTSILNDKEPMNIGLALSGWSQTDHRRHCEDAMQQNNNNGGSNIRIEEYVSSLIKNPDFSMSLAEAVVRTITGQQKQQDINLNLDLPEE*).

[0030] The present invention provides a recombinant vector for expressing the protein described in the above technical solution.

[0031] 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. The corresponding recombinant expression vector can be flexibly selected according to the type of host cell. In the embodiment of the present invention, the pCambia3300 plasmid is used as the backbone vector to illustrate the soybean GmWRKY40 The biological function of the gene. GmWRKY40 The cloning site of the gene is H ind Ⅲ The present invention has no special restrictions on the preparation method of the recombinant vector. The recombinant vector construction method known in the art can be used, for example, to amplify soybean enzyme recognition sites using primers with enzyme cleavage sites. GmWRKY40 Gene fragment or gene synthesis method to obtain soybean with enzyme cutting site GmWRKY40 The gene fragment was then subjected to restriction enzyme digestion along with the backbone vector using the same endonuclease, and the resulting linear vector and gene fragment were connected. The connection product was verified and the soybean was successfully cloned. 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 Expression levels of gene fragments.

[0032] The present invention provides a recombinant strain comprising the soybean GmWRKY40 gene or the recombinant vector.

[0033] In the present invention, the host strain of the recombinant strain includes Agrobacterium strain EHA105The present invention has no special restrictions on the construction method of the recombinant strain, and the recombinant strain can be transformed using methods well known in the art, such as electroporation of Agrobacterium or heat shock method. In the embodiment of the present invention, the recombinant strain constructed is soybean GmWRKY40 The gene was transferred into the host plant, and after 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.

[0034] 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:

[0035] Soybeans GmWRKY40 Gene construction recombinant expression vector;

[0036] The constructed recombinant expression vector is transferred into soybean to obtain transgenic soybean plants;

[0037] The transgenic soybean plants are grown and managed until maturity, and soybean seeds are harvested to obtain soybean seeds rich in soybean isoflavones.

[0038] 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.

[0039] In the present invention, the method for transferring the constructed recombinant expression vector into soybeans is preferably achieved through Agrobacterium-mediated transformation. Specifically, the Agrobacterium strain carrying the recombinant expression vector is infected with germinating soybean seeds, co-cultivated to obtain transformed seedlings, and screened for 7-10 days in a screening medium containing glufosinate (PPT) (125 mg / L). After elongation and rooting culture and hardening, the seedlings are subjected to molecular testing 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 of the T3 transgenic lines using primers 3300-WRKY40-486 and 3300-Bar-nos402 revealed that the expected bands could be detected, indicating that the GmWRKY40 gene had been successfully integrated into the soybean genome and could be inherited by offspring.

[0040] In the present invention, the soybean isoflavone content of the soybean seeds obtained from the T3 generation transgenic strains 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, and total isoflavones: 3231.34~3429.26μg / g, which are significantly improved compared with wild-type soybeans, with DZ, GC, GT and TI increasing by 34.59%, 13.14%, 26.2% and 25.15%, respectively.

[0041] 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.

[0042] 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.

[0043] The present invention provides the soybean GmWRKY40 Application of the gene, the protein, the recombinant vector or the recombinant strain in soybean breeding.

[0044] 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 richness in isoflavones means that compared with wild-type soybean plants, the isoflavone content in the seeds of transgenic soybean plants is significantly increased, with daidzein being 1043.94~1056.32μg / g, glycitein being 679.85~976.53μg / g, genistein being 1198.49~1705.47μg / g, and total isoflavones being 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.

[0045] 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 single-plant grain weight and 100-grain weight of soybeans compared with soybeans before overexpression. In the present invention, the 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 and the wild-type Williams 82, but the single-plant grain weight (17.1 g) and 100-grain weight (18.8 g) were significantly higher than those of the wild-type Williams 82.

[0046] In the present invention, the protein and oil ratio advantages in the soybean seeds refer to that the protein content ratio of the transgenic soybean seeds is significantly increased compared with the wild type, and the oil content ratio also has an increasing trend.

[0047] 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 number of main stem nodes of the transgenic soybean plant. The changes in flower development time include delayed flower development time.

[0048] Soybean provided by the present invention GmWRKY40 By stably overexpressing the gene in soybean plants, it is beneficial to greatly increase the isoflavone content of the grain and the protein and oil content of the soybean, 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.

[0049] 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.

[0050] The agarose and nucleic acid dye used in the present invention are provided by Lamblide.

[0051] The marker used—LD DSTM 2000—was purchased from Guangzhou Dongsheng Company;

[0052] The RNA extraction reagent RNAiso Plus was purchased from TaKaRa;

[0053] Restriction endonucleases were provided by Thermo Fisher Scientific;

[0054] The KOD One™ PCR Master Mix -Blue enzyme used for high-fidelity PCR amplification, the conventional 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.

[0055] The homologous recombination ligation kit ClonExpress II One Step Cloning Kit and the 2 × Rapid Taq Master Mix used for bacterial rapid PCR were purchased from Nanjing Novozymes Biotechnology Co., Ltd.

[0056] Gel recovery and plasmid extraction kits were provided by OMEGA;

[0057] The reagents used for soybean genetic transformation were purchased from Sigma;

[0058] The synthesis of gene primers and gene sequencing were completed by Beijing Ruibo Company.

[0059] The Agilent 1100 high performance liquid chromatography (HPLC) detection platform was used to quantitatively analyze the different components of soybean isoflavones in the test materials.

[0060] 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.

[0061] Unless otherwise specified, other experimental reagents and instruments can be purchased through commercial channels.

[0062] 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.

[0063] RNA was extracted using TaKaRa RNAiso Plus and reverse transcribed into cDNA using the ReverTra AceqPCR RT Master Mix with gDNA Remover reverse transcription kit for soybean cloning. GmWRKY40 Gene, primers synthesized by Beijing Ruibo Company were used to clone the target gene fragment using KOD OneTM PCR Master Mix -Blue-high-fidelity enzyme;

[0064] By restriction enzyme Hind Ⅲ The pCambia3300 vector was digested with a single enzyme.

[0065] Will GmWRKY40 The gene was constructed into an expression vector and transformed into DH5α competent Escherichia coli. 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 method to obtain stable transgenic plants.

[0066] Agilent 1100 HPLC detection platform was used to quantitatively analyze the different components of soybean isoflavones in the test materials.

[0067] Overexpression GmWRKY40 The gene can significantly increase the isoflavone content in seeds and improve the quality of soybeans. The invention can be used to develop new high-quality soybean varieties.

[0068] Example 1

[0069] GmWRKY40 Acquisition of genes

[0070] Using the high-isoflavone soybean variety Zhongdou 27 as the recipient parent and the low-isoflavone variety Jiunong 20 as the recurrent parent, a BC2F5 near-isogenic line population of 175 families was constructed through multi-generation backcrossing combined with marker screening. Genotyping was performed on each family within the population, and the ICIM-ADD method in the bip module of IciMapping v4.2 was used to map the QTL. It was found that qISO14-1 was associated with isoflavone content, and a candidate gene was identified within the 161 kb region of this QTL. GmWRKY40 The sequence variation of isoflavones was significantly associated with the content of Figure 1 ).

[0071] Example 2

[0072] change GmWRKY40 Construction of genetically modified soybean plants.

[0073] 1. GmWRKY40 Gene cloning

[0074] 1) Total RNA extraction (Trizol method)

[0075] ① Use scissors to cut the tender leaves of Zhongdou 27 and place them in a 2.0ml centrifuge tube. Quickly place the tube in a liquid nitrogen box for freezing and grinding. Add 1mL of pre-cooled Trisol extract to the centrifuge tube, shake thoroughly to mix, and let it stand on ice for 5 minutes.

[0076] ② Add 200 μL of chloroform, shake vigorously to mix thoroughly, let it stand for 3-5 minutes, and then centrifuge at 4°C and 12,000 rpm for 15 minutes;

[0077] ③ Remove the supernatant and transfer it to 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.

[0078] ④ Centrifuge at 4°C, 12,000 rpm for 10 min;

[0079] 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.

[0080] ⑥ Repeat step ⑤ once;

[0081] ⑦ Discard the supernatant, empty the tube for 2 minutes, remove the remaining supernatant with a pipette, and place on ice to dry for 10 minutes;

[0082] ⑧ After drying, add 20 μl DEPC water to dissolve and check the quality by gel electrophoresis ( Figure 2 Middle A).

[0083] 2) Total RNA reverse transcription

[0084] Total RNA was reverse transcribed into cDNA according to the TOYOBO cDNA synthesis kit (ReverTra Ace qPCR RT Master Mix with gDNA Remover, CAS No.: FSQ-301). The specific steps are as follows:

[0085] ① RNA denaturation: Place the RNA in a 65°C metal bath, keep warm for 5 minutes, and then cool it in ice.

[0086] ② Removal of genomic DNA: Use the ReverTra Ace qPCR RT Master Mix with gDNARemover Kit to remove residual genomic DNA. The reaction system is shown in Table 1:

[0087] Table 1 Genomic DNA removal reaction system and procedures

[0088]

[0089] 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.

[0090] Table 2 Reverse transcription reaction system and procedure

[0091]

[0092] Select the master copy gene sequence and design a pair of GmWRKY40 Primers were added to the 5' and 3' ends of the CDS region and appropriate restriction enzyme sites were introduced. GmWRKY40 Primers for gene cloning:

[0093] GmWRKY40 -F:TCGAGCTCCGTCGACAAGCTTATGGTCGTTCAACT(SE ID NO:3);

[0094] GmWRKY40 -R: GCCCTTGCTCACCATAAGCTTTCACTCTTCAGGAA (SE ID NO:4);

[0095] The cloning PCR reaction system was referred to the kit instructions. The PCR reaction conditions were: 98°C pre-denaturation for 3 minutes, 98°C denaturation for 10 seconds, 58°C annealing for 5 seconds, 68°C extension for 5 seconds, amplification for an appropriate number of times; 68°C final extension for 10 minutes, and the full-length CDS sequence of the gene was obtained (837 bp). Figure 2 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℃.

[0096] 2. GmWRKY40 Gene vector construction

[0097] Utilization of circular pCambia3300 plasmid hind Ⅲ The enzyme was digested by single enzyme and then separated by gel electrophoresis at 37℃ for 2~4h. Figure 3 ), the recovered target fragment was ligated with the linearized pCambia3300 vector fragment by homologous recombination to obtain a ligation product. The ligation system is shown in Table 3 with reference to the relevant instructions.

[0098] Table 3 Homologous recombination ligation reaction system and procedure

[0099]

[0100] The ligation product was transformed into DH5α competent cells, and the positive strain was sequenced correctly, indicating that pCambia3300 was successfully constructed. -GmWRKY40 The recombinant plasmid was then extracted and transformed into Agrobacterium EHA105. The Agrobacterium transformation method was as follows:

[0101] 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 an ice-water mixture, insert it into ice.

[0102] ② Add 0.01-1 μg of plasmid DNA per 100 μL competent medium, mix by hand by flicking the bottom of the tube, 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.

[0103] ③ Add 700 μL of antibiotic-free LB liquid medium and culture at 28°C with shaking for 2-3 hours.

[0104] ④ Harvest the bacteria by centrifugation at 6000 rpm for one minute. Collect about 100 μL of the supernatant and gently pipette to resuspend the bacteria. Spread the plate on an LB plate containing kanamycin and rifampicin antibiotics. Incubate the plate upside down at 28°C for 2-3 days and observe the growth of the plaque.

[0105] ⑤ After colonies have grown, pick a single plaque in a clean bench and inoculate it into 1 mL of LB liquid medium containing kanamycin (50 μg / mL) and rifampicin (20 μg / mL). Incubate the culture in a 28°C constant temperature shaker at 200 rpm for 24–48 h. Perform PCR verification of the bacterial solution.

[0106] ⑥ After successful PCR verification, the bacteria were stored.

[0107] The results showed that the recombinant plasmid pCambia3300- GmWRKY40 Successfully transformed into competent Agrobacterium tumefaciens EHA105 ( Figure 3 ).

[0108] 3. GmWRKY40 Genetic transformation

[0109] 1) Bacterial liquid preparation

[0110] A single colony of EHA105 carrying the target gene was picked and inoculated into LB liquid medium containing kanamycin (50 μg / mL) and rifampicin (20 μg / mL). The culture was incubated overnight at 28°C and 200 rpm. The bacterial solution was then transferred to a 100 ml breathable 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 use.

[0111] 2) Agrobacterium-mediated half-seed transformation method

[0112] ① Seed sterilization

[0113] Take mature Williams 82 soybean seeds with smooth, plump surfaces and no lesions, place them in a petri dish, and place them in a desiccator in a fume hood with the petri dish lid half open. Measure 96 ml of NaClO and place it in a beaker. Place the beaker in the middle of the petri dish. Once everything is ready, add 6 ml of concentrated hydrochloric acid (HCl) to the NaClO. Quickly close the desiccator lid. After sterilizing for 16-18 hours, remove the seeds and place them in a clean bench for 30 minutes to 1 hour to remove residual chlorine. Seal the lid and set aside.

[0114] ② Seed germination and transformation receptor preparation

[0115] In a clean bench, use tweezers to sow the sterilized seeds in a solid germination medium. When the seeds germinate and the cotyledons are about to break through the seed coat, use tweezers, a scalpel and other tools to remove the seed coat, retaining the hypocotyl 3 mm away from the cotyledons. Cut the cotyledons along the hypocotyl, gently scrape off the terminal buds and axillary buds between the cotyledons and the growth point with a scalpel, dip the seeds 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.

[0116] ③ Infection

[0117] The explants and the resuspension were placed in a sterile tissue culture bottle, sealed, and then placed in a 28°C constant temperature shaker at 100 rpm for infection for 30 min.

[0118] ④ Co-cultivation

[0119] 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, and the infected explants were placed with the cut side facing down on a culture dish containing CCM solid culture medium, and cultured in the dark in the tissue culture room for 3 days.

[0120] ⑤ Cultivation and screening of transformed seedlings

[0121] After co-cultivation, the cotyledonary node explants were washed three times with sterile water, blotted dry with sterile paper, and then spread flat on recovery medium with the cut surface facing downward for 7 days. The recovered cotyledonary node explants were then transferred to a selection medium containing glufosinate ammonium (PPT) (125 mg / L) for 7–10 days.

[0122] ⑥Elongation and rooting

[0123] Explants selected for PPT resistance were transferred to elongation medium and subcultured every 7 days. When the buds reached 3-4 cm in length, they were cut close to the base and transferred to rooting medium for rooting.

[0124] ⑦Seedling training

[0125] When the seedlings are strong enough, they are transferred to vermiculite and cultured under low light conditions for 3 days, and then transplanted to soil and cultured until maturity.

[0126] 4. GmWRKY40 Detection of genetically modified soybeans

[0127] 1) PCR (polymerase chain reaction) analysis of transgenic plants

[0128] DNA from transgenic and wild-type soybean Williams 82 was extracted according to the general method. PCR identification was performed using DNA as a template using specific detection primers and universal Bar primers designed based on the gene and vector sequences. The reaction system is shown in Table 4:

[0129] Table 4 PCR reaction system

[0130]

[0131] change GmWRKY40 Plant identification primers:

[0132] 3300- WRKY40 -486-F: 5'-AGCAAGTGCACAAAGCTTGA-3' (SEQ ID NO: 5);

[0133] 3300- WRKY40 -486-R: 5'-GCCAATGTTCATGGGTTCTT-3' (SEQ ID NO: 6);

[0134] 3300- Bar -nos402-F: 5'-GCGGTACCGGCAGGCTGAAG-3' (SEQ ID NO:7);

[0135] 3300- Bar -nos402-R: 5'-CCGCAGGAACCGCAGGAGTG-3' (SEQ ID NO: 8);

[0136] Reaction conditions: Bar Primer reaction program: 95℃ 3min; 35 cycles: 95℃ 15s, 54℃ 15s, 72℃ 15s; 72℃ 5min, store at 12℃. WRKY40 The reaction program for the -486 primer was as follows: 95°C for 3 minutes, followed by 35 cycles of: 95°C for 15 seconds, 58°C for 15 seconds, and 72°C for 15 seconds; then 72°C for 5 minutes, followed by storage at 12°C. After the reaction, 6 μL of the PCR product was analyzed by 2% agarose gel electrophoresis.

[0137] After PCR and Bar test strips testing, 17 T0 generation transgenic plants were obtained. The T0 generation positive plants were propagated in the greenhouse and used 3300- WRKY40 -486 primers and 3300- Bar -nos402 primers were used to detect the T3 transgenic lines by PCR ( 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.

[0138] 2) Analysis of target gene transcription levels in transgenic plants

[0139] ① Transfer GmWRKY40Fluorescence quantitative PCR of genetically modified soybean

[0140] A. Total RNA was extracted and reverse transcribed into cDNA. The cDNA was diluted to adjust the concentration to 300 ng / μl and used as a template for quantitative PCR.

[0141] B. Real-time fluorescence quantitative PCR

[0142] Transfer GmWRKY40 The cDNA of leaf tissue of gene plant was used as template and amplified by real-time fluorescence quantitative PCR to detect GmWRKY40 Gene expression, and internal reference gene Actin The average expression level of the target gene and the internal reference gene was used as a reference (three replicates were set for each treatment).

[0143] GmWRKY40 Gene fluorescence quantitative primers:

[0144] qGmWRKY40-F: 5'-TCCACATCTAAAGGCTCATCACCAG-3' (SEQ ID NO: 9);

[0145] qGmWRKY40-R: 5'-GCATGGCATCTTCACAATGTCTCC-3' (SEQ ID NO: 10);

[0146] Actin Gene fluorescence quantitative primers:

[0147] Actin4-F: 5'-GTTTCAAGCTCTTGCTCGTAATCA-3' (SEQ ID NO: 11);

[0148] Actin4-R: 5'-GTGTCAGCCATACTGTCCCCATTT-3' (SEQ ID NO: 12).

[0149] The reaction system is as follows: THUNDERBIRD ® Next SYBR ® qPCR Mix 10 μL; ddH2O 6.4 μL; upstream primer (10 μmol / L) 0.8 μL; downstream primer (10 μmol / L) 0.8 μL; cDNA template 2 μL. PCR program included: 95°C for 30 s; 95°C for 5 s, 60°C for 30 s, 72°C for 15 s, 40 cycles. The CT value in the relative quantification ΔΔCT method was the average of three replicates. The relative copy number was calculated using the formula 2. -ΔΔCT (ΔΔCT=CT 目的基因 - CT 内参Actin基因 ).

[0150] The present invention used 11 T3 generation PCR screening positive plants cDNA to do RT-qPCR detection. The results showed that the transgenic soybean GmWRKY40 The expression level is 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 obtained GmWRKY40 There were 4 transgenic plants with high gene expression, including the transgenic line GmWRKY40 The highest gene expression level was 53 times that of wild-type soybean ( Figure 5 ).

[0151] ② Transfer GmWRKY40 Semi-quantitative PCR detection of genetically modified soybean plants

[0152] Take 4 T3 generation positive plants young leaves and transfer them to GmWRKY40 The cDNA of the leaf tissue of the gene and the normal cultivated plant 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. Actin The average expression level of Figure 6 The primer sequences, system and reaction procedures were the same as those for the fluorescent quantitative PCR described above.

[0153] 3) Western-blot detection of transgenic plants

[0154] Protein extraction: Grind 0.3-0.5g of fresh leaves with liquid nitrogen until finely ground. Add 400µl of protein extraction buffer, mix thoroughly, vortex, and ice-bath for 10 minutes. Centrifuge at 13,000 rpm for 15 minutes at 4°C. Pipette the supernatant into a 1.5ml protease-free centrifuge tube. Add 80µl of this supernatant to 20µl of protein loading buffer, mix thoroughly, boil in boiling water for 10 minutes, and centrifuge at 12,000 rpm for 30 seconds. Pipette 20µl of the supernatant for spotting.

[0155] Prepare the gel: Align a clean glass plate and place it on a gel preparation rack. Pour the prepared separating 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 off the upper layer of distilled water and absorb it with filter paper. Add concentrated gel until it overflows, insert a comb, and let it gel for about 15 minutes.

[0156] Spotting: Add 1× running buffer to the electrophoresis tank, quickly pull out the comb vertically, and spot the sample vertically against the wall;

[0157] Gel running: Use 80V voltage for the stacking gel. After the sample runs into the separation gel, observe whether the marker runs away and adjust the voltage accordingly. Use 120V voltage for the separation gel. Select the electrophoresis time according to the protein size.

[0158] Remove the gel and transfer the membrane: Carefully pry up the small glass plate and remove the concentrated gel left on the large glass plate. Prepare two WB transfer sponge pads and one PVDF membrane, cut to the same size as the gel. Place the PVDF membrane in methanol in advance and soak the sponge pad in transfer solution in advance.

[0159] The order of membrane transfer from negative electrode to positive electrode is: sponge, glue, PVDF membrane, sponge. Each layer needs to be driven out of the air bubbles with a glass rod. Use 400mA current to transfer the membrane, which takes about 30 minutes.

[0160] Blocking: After transfer, take out the PVDF membrane and place it on a plate with blocking solution. Block at 60 rpm for 1 hour at room temperature.

[0161] Primary antibody reaction: Place the diluted primary antibody and PVDF membrane into a plate and shake at 60 rpm for 1 hour at room temperature;

[0162] Wash the membrane: Pour off the primary antibody in the dish, add 20 ml of TBST, shake at 60 rpm for 10 minutes at room temperature, repeat the previous step, pour off the TBST, add 20 ml of TBS and wash the membrane again for 10 minutes;

[0163] Secondary antibody reaction: Place the diluted secondary antibody and PVDF membrane into a plate and shake at 60 rpm for 1 hour at room temperature;

[0164] Membrane washing: the same as the above membrane washing operation;

[0165] development.

[0166] The gel electrophoresis of RT-PCR products showed that under the same conditions of internal reference amplification, no endogenous GmWRKY40 The wild-type plant (CK2) and samples 1-4 all showed endogenous GmWRKY40 The 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 the transgenic plants GmWRKY40 The gene was successfully overexpressed.

[0167] The 11 transgenic strains that were positive by quantitative PCR GmWRKY40 Genetically modified soybean plants were tested for BAR protein using Agene's PAT / BAR Gold Standard Rapid Immunoassay Kit ( Figure 7 A), and then the tag gene was detected by Western-Blot, and bands were detected in 8 strains ( Figure 7 (B in the figure shows some test results), indicating that the marker gene successfully encodes protein products in the transgenic plants.

[0168] Example 2

[0169] GmWRKY40 Identification of isoflavone content in genetically modified soybeans

[0170] 1) Soy isoflavone extraction

[0171] Harvest T3 transfection GmWRKY40 Genetically modified soybean plant seeds and dried soybean kernels were selected and ground into powder. Accurately weigh 0.1 g of sample into a 50 mL centrifuge tube, add 7 mL of 2 mol / L hydrochloric acid solution, vortex for 10 seconds to thoroughly mix, and then acid-immerse in a 90°C water bath for 90 minutes. After cooling to room temperature, add approximately 3.5 mL of 4 mol / L sodium hydroxide solution to a near-neutral pH. Then, add 80% anhydrous ethanol to a volume of 25 mL, invert to mix, and allow to stand for 30 minutes for extraction. The extracted liquid was filtered through a 0.22 μm syringe filter, transferred into a 1.5 mL liquid phase injection vial, and sealed. Store at 4°C until testing. Each sample was tested in triplicate.

[0172] 2) Qualitative and quantitative analysis of soy isoflavones

[0173] An Agilent 1100 high-performance liquid chromatography (HPLC) platform was used to quantitatively analyze the different components of soy isoflavones in the test materials. The following detection conditions were used: chromatographic-grade methanol and sterile deionized water were used as the mobile phase. The liquid phase system was first flushed with chromatographic-grade methanol. After the absorbance and pressure stabilized, the methanol:sterile water ratio was adjusted to a 1:1 ratio for constant elution. The flow rate was 1 mL / min, the column temperature was 35°C, the detection wavelength was 254 nm, and the injection volume was 10 μL. The approximate peak times of the individual isoflavone components were determined using isoflavone analytical standards, and the sample loading time was determined. The peak times of DZ, GC, and GT were 7 min 30 s, 8 min, and 11 min, respectively. The analysis time for a single sample was 15 min.

[0174] The content of each component of isoflavone was calculated by peak area method. GmWRKY40The 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 ).

[0175] Example 3

[0176] GmWRKY40 Identification of agronomic traits of transgenic soybeans

[0177] Investigation transfer GmWRKY40 The growth period and plant shape of the T3 generation of the transgenic strain were analyzed. After the plants matured, transgenic-positive plants were tested for biological yield (mainly including plant height, number of nodes, number of branches, number of pods, number of grains, and grain weight). Protein and oil content in transgenic and wild-type Williams 82 seeds was measured using a near-infrared grain analyzer.

[0178] As can be seen from Table 5, 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 turn GmWRKY40 There 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.

[0179] Table 5 Significance analysis of the difference in plant height between transgenic and wild-type soybeans

[0180]

[0181] Note: The data in the table are the average values ​​of 8 individual plants. # It indicates significant at the 0.05 level.

[0182] As can be seen from Table 6, GmWRKY40 There were no significant differences in the number of pods and single grains between the transgenic soybean plants and the wild-type Williams 82, but the single-plant grain weight and 100-grain weight were significantly higher than those of the wild-type Williams 82.

[0183] Table 6 Significance analysis of differences in grain weight, grain number and pod number between transgenic and wild-type soybeans

[0184]

[0185] Note: The data in the table are the average values ​​of 8 individual plants. # It indicates significant at the 0.05 level.

[0186] In order to compare the differences in soybean quality between transgenic and wild type, a near infrared grain analyzer was used to detect transgenic soybeans. GmWRKY40 Protein and oil content of genetically modified and wild-type Williams 82 seeds. Table 7 shows that the protein content of genetically modified soybean seeds is significantly higher than that of wild-type seeds, and the oil content also shows an increasing trend. GmWRKY40 The expression of the gene can increase the accumulation of protein and oil in soybean seeds.

[0187] Table 7 Significance analysis of differences in seed quality between transgenic and wild-type soybeans

[0188]

[0189] Note: The data in the table are the average values ​​of 6 individual plants. # It indicates significant at the 0.05 level.

[0190] From 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.

[0191] 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 the scope of protection of the present invention.

Claims

1. Soybeans GmWRKY40 Genes, soybeans GmWRKY40 Application of a gene-encoded protein, a recombinant vector or a recombinant strain expressing the protein in soybean breeding, wherein the soybean breeding includes increasing the isoflavone content in soybean seeds, increasing the protein content in soybean seeds and increasing soybean yield; The soybean GmWRKY40 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; The amino acid sequence of the protein is shown in SEQ ID NO: 2; The recombinant strain comprises the soybean GmWRKY40 gene or the recombinant vector.

2. The application according to claim 1, characterized in that The isoflavones include at least one of the following: daidzein, glycitein and genistein.

3. Soybeans GmWRKY40 Genes, soybeans GmWRKY40 Application of a gene-encoded protein, a recombinant vector or a recombinant strain expressing the protein in constructing transgenic soybeans rich in isoflavones; The soybean GmWRKY40 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; The amino acid sequence of the protein is shown in SEQ ID NO: 2; the recombinant strain contains the soybean GmWRKY40 gene or the recombinant vector.

4. A method for increasing the content of soybean isoflavones, characterized in that: The following steps are involved: Soybeans GmWRKY40 Gene construction recombinant expression vector; The constructed recombinant expression vector is transferred into soybean to obtain transgenic soybean plants; Growing and managing the transgenic soybean plants until maturity, harvesting soybean grains to obtain isoflavone-rich soybean grains; The soybean GmWRKY40 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

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