Application of transcription factor GmMYB77 and its encoding gene in regulating soybean isoflavone content

Abstract

The present invention belongs to the field of plant genetic engineering and specifically relates to the use of the transcription factor GmMYB77 and its encoding gene in regulating the content of soybean isoflavones. A method for regulating the content of isoflavones in plants is provided, comprising: reducing the level of the transcription factor GmMYB77 in the plant to increase the isoflavone content of the plant, or increasing the level of the transcription factor GmMYB77 in the plant to reduce the isoflavone content of the plant; the amino acid sequence of the transcription factor GmMYB77 is shown in SEQ ID NO: 2. The transcription factor GmMYB77 negatively regulates the content of soybean isoflavones by inhibiting the gene expression of key enzymes in the soybean isoflavone biosynthesis pathway.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and particularly relates to the application of transcription factor GmMYB77 and its encoding gene in regulating the content of soybean isoflavones. Background Art

[0002] Soy isoflavones are secondary metabolites formed during soybean growth and belong to the flavonoid class of compounds. Soy isoflavones are a class of non-steroidal substances with high nutritional value and health benefits. Due to their structural and functional similarities to estrogen, they are also known as phytoestrogens. Soy isoflavones primarily consist of 12 components, including three aglycones (daidzein, glycitein, and genistein) and nine corresponding glycosides (daidzein, glycitein, genistin, malonyl daidzein, malonyl glycitein, malonyl genistin, acetylated daidzein, acetylated glycitein, and acetylated genistin). Glycitein and its corresponding glycoside forms impart a bitter taste in soy products, with malonyl glycitein and acetylated glycitein having the lowest bitterness threshold. Studies have shown that soy isoflavones play an important role in anti-cancer, improving osteoporosis, reducing cardiovascular and cerebrovascular disease, and preventing and curing menopausal syndrome in women. In plants, isoflavones possess antifungal, antibacterial, antiviral, and antioxidant properties, and they act as signaling molecules to induce soybean nodulation. Due to their significant application value in food and health supplements, soy isoflavones have garnered widespread attention. Therefore, cultivating high-isoflavone soybean varieties for specialized applications is a major goal of soybean breeding for nutritional quality.

[0003] Genetic engineering is an effective means of improving crop traits. Modifying key enzyme genes in the isoflavone biosynthesis pathway can increase the isoflavone content in soybeans. GmMYB77 is an R2R3 MYB transcription factor in soybean. The gene encoding it, GmMYB77, is assigned the gene ID Glyma.04G036700 in the soybean genome version Glycine max Wm82.a2.v1. Currently, no functional information on GmMYB77 has been reported. Summary of the Invention

[0004] The present invention aims to cultivate new soybean varieties with different isoflavone contents.

[0005] The present invention provides a method for regulating the isoflavone content in plants, which comprises: reducing the level of transcription factor GmMYB77 in the plant to increase the isoflavone content in the plant, or increasing the level of transcription factor GmMYB77 in the plant to reduce the isoflavone content in the plant; the amino acid sequence of the transcription factor GmMYB77 is shown in SEQ ID NO: 2.

[0006] In the above method, the level of the transcription factor GmMYB77 in the plant can be reduced by silencing or knocking out the gene encoding the transcription factor GmMYB77 in the plant, or the level of the transcription factor GmMYB77 in the plant can be increased by overexpressing the gene encoding the transcription factor GmMYB77 in the plant.

[0007] In the above method, the nucleotide sequence of the gene encoding the transcription factor GmMYB77 is shown in SEQ ID NO: 1.

[0008] In the above method, the CRISPR / Cas9 system can be used to knock out the gene encoding the transcription factor GmMYB77 in plants.

[0009] In the above method, the target sequence of the gene encoding the transcription factor GmMYB77 used in the CRISPR / Cas9 system can be the nucleotide sequence shown in SEQ ID NO:9.

[0010] In the above method, the gene encoding the transcription factor GmMYB77 in the plant can be knocked out by introducing the gene encoding the sgRNA targeting the target sequence and the gene encoding Cas9 into the plant.

[0011] In the above method, the plant may be soybean.

[0012] The present invention also provides a product for increasing the isoflavone content in soybeans, which is used to reduce the level of transcription factor GmMYB77 in soybeans, wherein the amino acid sequence of the transcription factor GmMYB77 is shown in SEQ ID NO: 2.

[0013] The product may be a reagent and / or instrument required for site-directed editing of the gene encoding the transcription factor GmMYB77 using the CRISPR / Cas9 system.

[0014] The reagent can be reagent 1, reagent 2, reagent 3, reagent 4 or reagent 5; the reagent 1 is an sgRNA targeting the target sequence shown in SEQ ID NO: 9; the reagent 2 is a composition of the reagent 1 and Cas9; the reagent 3 is a recombinant vector containing the coding gene of reagent 1; the reagent 4 is a composition composed of the reagent 3 and a recombinant vector containing the coding gene of Cas9; the reagent 5 is a recombinant vector containing the coding gene of reagent 1 and the coding gene of Cas9.

[0015] Experiments have shown that the transcription factor GmMYB77 negatively regulates soybean isoflavone content by inhibiting the expression of isoflavone synthase 1 (IFS1), isoflavone synthase 2 (IFS2), chalcone synthase 7 (CHS7), chalcone synthase 8 (CHS8), and isoflavone O-methyltransferase 1 (IOMT1) genes in the soybean isoflavone biosynthesis pathway. Overexpression of GmMYB77 in soybean hairy roots significantly reduced total isoflavone content, while inhibition of GmMYB77 significantly increased it. In soybean plants, overexpression of the GmMYB77 gene can significantly reduce the isoflavone content in seeds, especially the content of malonyl glycitin; knocking out the GmMYB77 gene can significantly increase the isoflavone content in seeds, especially the content of malonyl glycitin. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the structure of the GmMYB77 gene overexpression vector pGFPGUSplus-GmMYB77.

[0017] Figure 2 Schematic diagram of the structure of the GmMYB77 gene silencing vector pGFPGUSplus-GmMYB77-RNAi.

[0018] Figure 3 The relative expression levels of GmMYB77 gene and isoflavone contents in soybean hairy roots are shown; (a) Relative expression level of GmMYB77 gene in GmMYB77 overexpressing hairy roots; (be) Contents of daidzein, malonyl daidzein, malonyl glycitin and total isoflavones in GmMYB77 overexpressing hairy roots; (f) Relative expression level of GmMYB77 gene in GmMYB77 silenced hairy roots; (gj) Contents of daidzein, malonyl daidzein, malonyl glycitin and total isoflavones in GmMYB77 silenced hairy roots; Empty vector indicates the vector containing an empty vector. Hairy roots are the control group; GmMYB77-OE-1, GmMYB77-OE-2 and GmMYB77-OE-3 represent hairy roots with GmMYB77 overexpression; GmMYB77-RNAi-1, GmMYB77-RNAi-2 and GmMYB77-RNAi-3 represent hairy roots with GmMYB77 silenced; one asterisk (*) indicates that the data in this group are significantly different from those in the control group (p<0.05); two asterisks (**) indicate that the data in this group are very significantly different from those in the control group (p<0.01).

[0019] Figure 4 Schematic diagram of the structure of the GmMYB77 gene overexpression vector pTF101-GmMYB77-GFP.

[0020] Figure 5 Schematic diagram of the structure of the signal vector constructed for tobacco transient expression experiments, where promoter represents the natural promoter pGmIFS1, pGmIFS2, pGmCHS7 or pGmCHS8.

[0021] Figure 6 Figure 3 shows the regulation of the activity of the natural promoters of key enzyme genes in the isoflavone biosynthesis pathway by the transcription factor GmMYB77; (a) Schematic diagram of the tobacco transient expression vector; (b) GmMYB77 regulates the activity of the natural promoters of the GmIFS1, GmIFS2, GmCHS7, and GmCHS8 genes, where two asterisks (**) indicate that there are very significant differences between the data in this group and the control group (p<0.01).

[0022] Figure 7 Schematic diagram of the structure of the GmMYB77 gene editing vector GmMYB77-CRISPR / Cas9.

[0023] Figure 8 The test results of soybean GmMYB77 overexpression lines; (a) BAR test strip test results of GmMYB77 overexpression lines; (b) relative expression level of GmMYB77 gene in GmMYB77 overexpression lines, where two asterisks (**) indicate that there is a very significant difference between the data of this group and the control group data (p<0.01); (c) Western Blot test results of GFP protein in GmMYB77 overexpression lines; GmMYB77-1, GmMYB77-2, and GmMYB77-4 represent three GmMYB77 overexpression lines; TL1 represents "Tianlong No. 1" soybean, which serves as the control group.

[0024] Figure 9The figures are the phenotypic analysis results of soybean GmMYB77 overexpression lines; (ac) isoflavone content in seeds of GmMYB77 overexpression lines; (dh) relative expression levels of GmIFS1, GmIFS2, GmCHS7, GmCHS8 and GmIOMT1 genes in leaves of GmMYB77 overexpression lines; GmMYB77-1, GmMYB77-2 and GmMYB77-4 represent three GmMYB77 overexpression lines; TL1 represents "Tianlong No. 1" soybean, which is the control group; one asterisk (*) indicates that the p-value is less than 0.05, indicating that there is a significant difference between the data of this group and the data of the control group; two asterisks (**) indicate that the p-value is less than 0.01, indicating that there is a very significant difference between the data of this group and the data of the control group.

[0025] Figure 10 Screening results of CRISPR / Cas9-mediated soybean GmMYB77 knockout plants; (a) Position and sequence of gRNA in GmMYB77; (b) Relative expression level of GmMYB77 gene in GmMYB77 knockout plants, where two asterisks (**) indicate that there is a very significant difference between the data in this group and the control group (p<0.01); (c) PCR detection results of Bar gene in GmMYB77 knockout plants; (d) Test strip detection results of Bar gene in GmMYB77 knockout plants; Gmmyb77-1, Gmmyb77-2 and Gmmyb77-3 represent three Gmmyb77 homozygous knockout materials; TL1 represents "Tianlong No. 1" soybean, which serves as the control group.

[0026] Figure 11 Figure 3 Phenotypic identification of soybean GmMYB77 knockout plants; (ac) isoflavone content in seeds of GmMYB77 knockout plants; (dh) relative expression levels of GmIFS1, GmIFS2, GmCHS7, GmCHS8 and GmIOMT1 genes in leaves of GmMYB77 knockout plants; Gmmyb77-1, Gmmyb77-2 and Gmmyb77-3 are GmMYB77 homozygous knockout materials; TL1 represents "Tianlong No. 1" soybean, which serves as the control group; one asterisk (*) indicates a p-value less than 0.05, indicating that there is a significant difference between the data in this group and the data in the control group; two asterisks (**) indicate a p-value less than 0.01, indicating that there is a very significant difference between the data in this group and the data in the control group. DETAILED DESCRIPTION

[0027] The following examples are provided:

[0028] 1. Use of the transcription factor GmMYB77 or its encoding gene in regulating the isoflavone content in plants, wherein the amino acid sequence of the transcription factor GmMYB77 is shown in SEQ ID NO: 2.

[0029] 2. The use described in Example 1, comprising: reducing the level of the transcription factor GmMYB77 in the plant to increase the isoflavone content in the plant, or increasing the level of the transcription factor GmMYB77 in the plant to reduce the isoflavone content in the plant.

[0030] 3. The use described in Example 2, wherein the level of the transcription factor GmMYB77 in the plant is reduced by silencing or knocking out the gene encoding the transcription factor GmMYB77 in the plant, or the level of the transcription factor GmMYB77 in the plant is increased by overexpressing the gene encoding the transcription factor GmMYB77 in the plant.

[0031] 4. The application described in Example 3, wherein a silencing vector of the gene encoding the transcription factor GmMYB77 is constructed using the plant expression vector pGFPGUSplus, and the silencing vector is introduced into the plant through Agrobacterium-mediated genetic transformation technology, thereby reducing the level of the transcription factor GmMYB77 in the plant.

[0032] 5. The application described in Example 3, wherein the gene editing vector RPS5A::Cas9 is used to construct a knockout vector of the gene encoding the transcription factor GmMYB77, and the knockout vector is introduced into the plant through Agrobacterium-mediated genetic transformation technology, thereby reducing the level of the transcription factor GmMYB77 in the plant.

[0033] 6. The application described in Example 3, wherein the plant expression vector pGFPGUSplus is used to construct an overexpression vector of the gene encoding the transcription factor GmMYB77, and the overexpression vector is introduced into the plant through Agrobacterium-mediated genetic transformation technology, thereby increasing the level of the transcription factor GmMYB77 in the plant.

[0034] 7. The use described in Example 3, wherein the plant expression vector pTF101-GFP is used to construct an overexpression vector of the gene encoding the transcription factor GmMYB77, and the overexpression vector is introduced into the plant through Agrobacterium-mediated genetic transformation technology, thereby increasing the level of the transcription factor GmMYB77 in the plant.

[0035] 8. The use according to any one of Examples 1-7, wherein the nucleotide sequence of the gene encoding the transcription factor GmMYB77 is as shown in SEQ ID NO: 1.

[0036] 9. The use of any one of Examples 1-8, wherein regulating the isoflavone content in the plant comprises regulating the isoflavone content in the roots, stems, leaves, flowers, fruits and seeds of the plant.

[0037] 10. The use according to any one of embodiments 1-9, wherein the plant is soybean.

[0038] 11. A method for regulating the isoflavone content in a plant, comprising: reducing the level of transcription factor GmMYB77 in the plant to increase the isoflavone content in the plant, or increasing the level of transcription factor GmMYB77 in the plant to reduce the isoflavone content in the plant; the amino acid sequence of the transcription factor GmMYB77 is shown in SEQ ID NO: 2.

[0039] 12. The method of embodiment 11, wherein the level of the transcription factor GmMYB77 in the plant is reduced by silencing or knocking out the gene encoding the transcription factor GmMYB77 in the plant, or the level of the transcription factor GmMYB77 in the plant is increased by overexpressing the gene encoding the transcription factor GmMYB77 in the plant.

[0040] 13. The method described in Example 12, wherein a silencing vector encoding the gene of the transcription factor GmMYB77 is constructed using the plant expression vector pGFPGUSplus, and the silencing vector is introduced into the plant through Agrobacterium-mediated genetic transformation technology, thereby reducing the level of the transcription factor GmMYB77 in the plant.

[0041] 14. The method described in Example 12, wherein the gene editing vector RPS5A::Cas9 is used to construct a knockout vector of the gene encoding the transcription factor GmMYB77, and the knockout vector is introduced into the plant through Agrobacterium-mediated genetic transformation technology, thereby reducing the level of the transcription factor GmMYB77 in the plant.

[0042] 15. The method described in Example 12, wherein the plant expression vector pGFPGUSplus is used to construct an overexpression vector of the gene encoding the transcription factor GmMYB77, and the overexpression vector is introduced into the plant through Agrobacterium-mediated genetic transformation technology, thereby increasing the level of the transcription factor GmMYB77 in the plant.

[0043] 16. The method described in Example 12, wherein the plant expression vector pTF101-GFP is used to construct an overexpression vector of the gene encoding the transcription factor GmMYB77, and the overexpression vector is introduced into the plant through Agrobacterium-mediated genetic transformation technology, thereby increasing the level of the transcription factor GmMYB77 in the plant.

[0044] 17. The method described in any one of Examples 12-16, wherein the nucleotide sequence of the gene encoding the transcription factor GmMYB77 is shown in SEQ ID NO: 1.

[0045] 18. The method of any one of embodiments 12-17, wherein the gene encoding the transcription factor GmMYB77 is knocked out in the plant using the CRISPR / Cas9 system.

[0046] 19. The method described in any one of Examples 12-18, wherein the target sequence of the gene encoding the transcription factor GmMYB77 used in the CRISPR / Cas9 system is shown in SEQ ID NO: 9.

[0047] 20. The method of any one of embodiments 12-19, wherein the gene encoding the transcription factor GmMYB77 in the plant is knocked out by introducing a gene encoding an sgRNA targeting the target sequence and a gene encoding Cas9 into the plant.

[0048] 21. The method of any one of embodiments 11-20, wherein regulating the isoflavone content in the plant comprises regulating the isoflavone content in the roots, stems, leaves, flowers, fruits and seeds of the plant.

[0049] 22. The method of any one of embodiments 11-21, wherein the plant is soybean.

[0050] 23. A product for regulating the isoflavone content in soybeans, wherein the product is used to reduce the level of transcription factor GmMYB77 in soybeans, wherein the amino acid sequence of the transcription factor GmMYB77 is shown in SEQ ID NO: 2.

[0051] 24. The product of Example 23, wherein the product is a reagent and / or instrument required for site-directed editing of the gene encoding the transcription factor GmMYB77 using the CRISPR / Cas9 system.

[0052] 25. The product of Example 24, wherein the reagent is reagent 1, reagent 2, reagent 3, reagent 4, or reagent 5; the reagent 1 is an sgRNA targeting the target sequence shown in SEQ ID NO: 9; the reagent 2 is a composition consisting of the reagent 1 and Cas9; the reagent 3 is a recombinant vector containing the gene encoding the reagent 1; the reagent 4 is a composition consisting of the reagent 3 and a recombinant vector containing the gene encoding the Cas9; and the reagent 5 is a recombinant vector containing the gene encoding the reagent 1 and the gene encoding the Cas9.

[0053] The present invention will be further described below with reference to experimental examples. It should be understood that the following experimental examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0054] Unless otherwise specified, the reagents used in the following experimental examples are all conventional reagents in the art, commercially available or prepared according to conventional methods in the art, and the specifications are laboratory pure grade. Unless otherwise specified, the experimental methods and conditions used in the following experimental examples are all conventional experimental methods and conditions in the art, and reference can be made to relevant experimental manuals, known documents or manufacturer specifications. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0055] Plants used in the following experiments: Soybean (Glycine max) variety "Tianlong No. 1" (abbreviated as TL1), approved by the National Agricultural Science Institute 2008023, bred by the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences. Nicotiana benthamiana was grown in this laboratory.

[0056] The cells used in the following experiments: Agrobacterium rhizogenes K599 competent cells, Escherichia coli DH5α competent cells, EHA105 Agrobacterium competent cells, and ENA105 (pSoup) competent cells were purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd. Trans-T1 Escherichia coli competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0057] The main reagents used in the following experimental examples are: KOD FX high-fidelity enzyme (TOYOBO), product number KFX-101. BAR rapid test strips were purchased from AoChuang Biotechnology Co., Ltd., product number A07-13-413. B5 medium basal salts and corresponding vitamins (product number G398), MS medium basal salts and corresponding vitamins, and MSB medium (product number M519) were all purchased from Beijing Ximeijie Technology Co., Ltd. The plant genomic DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., product number DP321. The plant RNA extraction kit was purchased from Beijing Jinbaite Biotechnology Co., Ltd., product number ET111-01. The reverse transcription kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd., product number AT311-02. The DNA agarose gel recovery kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., product number DP209. In-fusion ligase was purchased from Baoriyi Biotechnology (Beijing) Co., Ltd., product number 639649. The protein rapid extraction kit was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd. with the product number CW0885M. The Dual-Glo Luciferase Assay System kit was purchased from Promega (Beijing) Biotechnology Co., Ltd. with the product number E1910.

[0058] The main instruments used in the following experimental examples are: PCR amplifier (Bio-RAD), electrophoresis apparatus (DYY-6C), desktop constant temperature oscillator (THZ-D), high-speed refrigerated centrifuge (SiGMR), high-speed desktop centrifuge (Sigma 3-30K), gel imaging analyzer (Tanon 3500), constant temperature incubator (LRH-250A), handheld fluorimeter and Bio-Rad MyiQ single-color fluorescence real-time quantitative PCR instrument, LUYOR-3260RB flashlight fluorescent protein observation microscope and Bio-RadMyiQ single-color fluorescence real-time quantitative PCR instrument, and SYNERGY H1 full-function microplate reader.

[0059] Table 1: PCR primers used in the following experiments

[0060]

[0061]

[0062] Experimental Example 1

[0063] Overexpression and silencing of the GmMYB77 gene in soybean hairy roots

[0064] 1.1 Gene cloning

[0065] RNA was extracted from the first true leaf of the soybean cultivar "Tianlong No. 1" (TL1) using a plant RNA extraction kit and reverse transcribed into cDNA using a reverse transcription kit. The resulting cDNA was used as a template to clone the complete coding region of the transcription factor GmMYB77 by polymerase chain reaction (PCR) using primers GmMYB77-F and GmMYB77-R (Table 1) for subsequent vector construction. The PCR system consisted of 25 μL 2× Mix, 1 μL F primer, 1 μL R primer, 2 μL template, and 21 μL sterile water. The PCR program was as follows: first, pre-denaturation at 98°C for 30 seconds; second, denaturation at 98°C for 10 seconds; third, annealing at 57°C for 10 seconds; fourth, extension at 68°C for 1 minute; 30 cycles from steps 2 to 4; and fifth, complete extension at 68°C for 10 minutes. The amino acid sequence of the transcription factor GmMYB77 is shown in SEQ ID NO: 2, encoded by the GmMYB77 gene. The coding sequence of the GmMYB77 gene is shown in SEQ ID NO: 1.

[0066] 1.2 Vector construction

[0067] The GmMYB77-OE fragment was amplified by PCR using the complete coding region cDNA of GmMYB77 as a template using primers GmMYB77-OE-F and GmMYB77-OE-R (Table 1). The PCR system consisted of 25 μL of 2× Mix, 1 μL of primer F, 1 μL of primer R, 2 μL of template, and 21 μL of sterile water. The PCR program was as follows: first, pre-denaturation at 98°C for 30 seconds; second, denaturation at 98°C for 10 seconds; third, annealing at 57°C for 10 seconds; fourth, extension at 68°C for 1 minute; 30 cycles from steps 2 to 4; and fifth, complete extension at 68°C for 10 minutes.

[0068] The GmMYB77-RNAi fragment was amplified by PCR using the complete GmMYB77 coding region cDNA as a template using primers GmMYB77-RNAi-F and GmMYB77-RNAi-R (Table 1). The PCR system consisted of 25 μL of 2× Mix, 1 μL of primer F, 1 μL of primer R, 2 μL of template, and 21 μL of sterile water. The PCR program was as follows: first, pre-denaturation at 98°C for 30 seconds; second, denaturation at 98°C for 10 seconds; third, annealing at 57°C for 10 seconds; fourth, extension at 68°C for 1 minute; 30 cycles from steps 2 to 4; and fifth, complete extension at 68°C for 10 minutes.

[0069] Using the commercially available plant expression vector pGFPGUSplus as a starting vector, a GmMYB77 gene overexpression vector and a GmMYB77 gene silencing vector were constructed, respectively. The pGFPGUSplus vector was double-digested with BglⅡ and BstEⅡ restriction enzymes to obtain a linearized pGFPGUSplus vector. The digestion system consisted of 5 μL of 10× buffer, 15 μL of pGFPGUSplus vector, 2 μL of BglⅡ endonuclease, 2 μL of BstEⅡ endonuclease, and 26 μL of sterile water. Digestion conditions were 37°C for 2 h.

[0070] The GmMYB77-OE fragment was introduced into the pGFPGUSplus vector by ligation to obtain the GmMYB77 gene overexpression vector, which was named pGFPGUSplus-GmMYB77 ( Figure 1 The ligation system consisted of 2 μL of 5× In-fusion, 4 μL of linearized pGFPGUSplus vector, 2 μL of the GmMYB77-OE fragment, and 2 μL of sterile water. Ligation conditions were: 50°C for 20 min.

[0071] The GmMYB77-RNAi fragment was introduced into the pGFPGUSplus vector by ligation to obtain the GmMYB77 gene silencing vector, which was named pGFPGUSplus-GmMYB77-RNAi ( Figure 2 The ligation system consisted of 2 μL of 5× In-fusion, 4 μL of linearized pGFPGUSplus vector, 2 μL of the GmMYB77-RNAi fragment, and 2 μL of sterile water. Ligation conditions were: 50°C for 20 min.

[0072] 1.3 Agrobacterium transformation

[0073] The pGFPGUSplus empty vector, pGFPGUSplus-GmMYB77, and pGFPGUSplus-GmMYB77-RNAi were transformed into Agrobacterium rhizogenes K599 competent cells by electroporation. The transformation method was as follows: add the plasmid to 100 μL of freshly thawed K599 competent cells, gently mix, and transfer the cells to an electroporation cuvette for transformation. Immediately after electroporation, add 500 μL of antibiotic-free YEP liquid medium to the cells, pipette 2-3 times to mix thoroughly, transfer the mixture to a 1.5 mL centrifuge tube, and incubate on a shaker at 200 rpm at 28°C for 3 hours. An appropriate amount of the bacterial culture was spread onto a YEP solid plate containing 50 μg / mL kanamycin and incubated at 28°C for 36-48 hours. Single clones were picked and identified by colony PCR using primers GFP-JC-F and GFP-JC-R (Table 1). The positive clones were sequenced to obtain recombinant Agrobacterium K599-pGFPGUSplus, K599-pGFPGUSplus-GmMYB77 and

[0074] K599-pGFPGUSplus-GmMYB77-RNAi.

[0075] 1.4 Hairy root induction culture

[0076] (a) Seed sterilization: Place healthy "Tianlong No. 1" soybean seeds in a petri dish. Add 80 mL of sodium hypochlorite and 5 mL of concentrated hydrochloric acid to a beaker. Place the petri dish and beaker in a sealed desiccator and sterilize the soybean seeds using chlorine gas for approximately 16-18 hours.

[0077] (b) Seed germination: The sterilized soybean seeds in step (a) were planted in a germination medium and germinated at 25° C. with 16 h of light and 8 h of darkness per day.

[0078] (c) Preparation of bacterial culture: Recombinant Agrobacterium K599-pGFPGUSplus, K599-pGFPGUSplus-GmMYB77, and K599-pGFPGUSplus-GmMYB77-RNAi were inoculated into fresh YEP liquid medium and cultured at 28°C with shaking at 200 rpm until the OD 600nmThe activated bacterial solution was inoculated into fresh YEP liquid medium and cultured at 28°C and 200 rpm until the OD 600nm is 0.6-0.8, and the second activated bacterial solution is obtained.

[0079] (d) Explant Preparation: Take the soybean seeds germinated 4-7 days after step (b), cut them 1-2 mm from the hypocotyl, split the cotyledons in half, remove the terminal buds, and gently make 5-7 cuts at the cotyledonary nodes with a razor blade to obtain explants.

[0080] (e) Explant infection: Take the second activated bacterial solution in step (c), centrifuge it at 6000 rpm for 10 min at room temperature, remove the supernatant, resuspend the bacteria in liquid co-culture medium, and adjust the OD value of the suspension. 600nm to 0.6-0.8 to obtain an infection solution; and infect the explant obtained in step (d) with the infection solution for 15-20 minutes.

[0081] (f) Co-cultivation: The infected explants were transferred to solid co-cultivation medium covered with sterile filter paper and cultured at 25°C in the dark for 3 days.

[0082] (g) Root induction culture: After co-cultivation, the explants were washed 3-5 times with liquid co-cultivation medium, and then transferred to root induction medium and cultured at 25°C with 16 h of light and 8 h of darkness per day.

[0083] (h) Hairy root detection: After root induction, explants were placed under a fluorescence microscope for observation. Hairy roots showing green fluorescence in the field of view were considered positive roots. Hairy roots containing the pGFPGUSplus empty vector (empty vector hairy roots), GmMYB77-overexpressing hairy roots, and GmMYB77-silenced hairy roots were obtained.

[0084] The formula of the above-mentioned germination medium is as follows: 3.21 g / L B5 medium, 20 g / L sucrose, adjust the pH to 5.8 with NaOH (1 M), add 7 g / L agar, sterilize at 120°C for 20 min, wait until the medium temperature drops to 50°C, add 1 mL / L B5 organic salt to the medium, mix well, and pour into a disposable plastic dish.

[0085] The formula of the above-mentioned liquid co-culture medium is as follows: 0.44 g / L 1 / 2MS inorganic salt, 20 g / L sucrose, 3.9 g / L methyl ethyl sulfonate (MES), adjusted to pH 5.4, sterilized at 120°C for 20 min, and after the culture medium temperature dropped to 50°C, 1 mL / L MS organic salt, 154 mg / L dithiothreitol (DTT), and 0.02 g / L acetosyringone (AS) were added, mixed, and poured into a disposable plastic dish.

[0086] The solid co-culture medium has the following formula: 0.44 g / L 1 / 2 MS inorganic salts, 20 g / L sucrose, 3.9 g / L methyl ethyl sulfonate (MES), adjusted to pH 5.4, added with 7 g / L agar, and sterilized at 120°C for 20 min. After the culture medium temperature drops to 50°C, 1 mL / L MS organic salts, 154 mg / L dithiothreitol (DTT), and 0.02 g / L acetosyringone (AS) are added, mixed, and poured into a disposable plastic dish.

[0087] The formula of the root induction medium is as follows: 2.2 g / L 1 / 2 MS medium, 20 g / L sucrose, 0.6 g / L MES, adjusted to pH 5.8, 7 g / L agar, sterilized at 120°C for 20 min, and after the medium temperature dropped to 50°C, 1 mL / L MS organic salt, 250 mg / L cefixime, and 250 mg / L carbenicillin were added. The mixture was mixed thoroughly and poured into a disposable plastic dish.

[0088] 1.5 Analysis of mMYB77 gene expression

[0089] RNA was extracted from hairy roots expressing an empty vector, overexpressing GmMYB77, and silenced GmMYB77, and reverse-transcribed into cDNA. Real-time quantitative PCR was performed using TaKaRa's SYBR Premix Ex Taq II using cDNA as a template to determine the relative expression of the GmMYB77 gene in soybean hairy roots. The reference gene was GmActin-6 (GenBank accession number: LOC100792119), whose nucleotide sequence is shown in SEQ ID NO: 3. Primers qGmMYB77-F and qGmMYB77-R (Table 1) were used to amplify the GmMYB77 gene. Primers qGmActin-F and qGmActin-R (Table 1) were used to amplify the GmActin-6 gene. The reaction system consisted of 5 μL 2× Mix, 0.4 μL F primer, 0.4 μL R primer, 1 μL cDNA, and 3.2 μL sterile water. The reaction procedure was as follows: first step, pre-denaturation at 98°C for 3 min; second step, denaturation at 98°C for 10 s; third step, annealing at 57°C for 1 min; fourth step, extension at 72°C for 1 min; 30 cycles from the second to the fourth step; fifth step, complete extension at 72°C for 3 min. Data were analyzed using Excel software and 2 -ΔΔCT The relative expression level of GmMYB77 gene was calculated by the method.

[0090] like Figure 3As shown in (a), the relative expression level of the GmMYB77 gene in GmMYB77-overexpressing hairy roots (GmMYB77-OE-1, GmMYB77-OE-2, and GmMYB77-OE-3) increased by 16.02 to 19.14 times compared with that in the hairy roots containing the empty vector.

[0091] like Figure 3 As shown in (f), compared with the hairy roots containing the empty vector, the relative expression level of the GmMYB77 gene in the GmMYB77-silenced hairy roots (GmMYB77-RNAi-1, GmMYB77-RNAi-2, and GmMYB77-RNAi-3) decreased by 43.5% to 52.7%.

[0092] 1.6 Determination of isoflavone content

[0093] Hairy roots expressing an empty vector, overexpressing GmMYB77, or silencing GmMYB77 were collected for isoflavone extraction and assayed. The isoflavone extraction and assay methods were as follows: The tissue to be tested was ground into a powder using a cyclone mill (mortar). 0.02 g of the powder was weighed into a 2 mL centrifuge tube. 1 mL of an extraction solution containing 70% (v / v) ethanol and 0.1% (v / v) acetic acid was added, and the centrifuge tube was shaken for 12 hours. The mixed extract was centrifuged at 2700 g for 10 minutes at 4°C. The supernatant was collected and filtered through a 0.2 μm filter (YMC, Kyoto, Japan). Isoflavone content was determined using an Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, California, USA). Quantitative analysis was performed using a YMC ODS AM-303 column (250 mm × 4.6 mm ID, S-5 μm, 120 Å). Mobile phase A consisted of 0.1% (v / v) acetic acid in water, and mobile phase B consisted of acetonitrile. The solvent flow rate was 1.0 mL / min, the injection volume was 10 μL, and a 70-minute linear gradient from 13% to 30% (v / v) acetonitrile was used. The UV detector wavelength was set at 260 nm, and the column temperature was set at 35°C. The soy isoflavone standard sample consists of 12 components, including daidzein, glycitein, genistein, daidzein, glycitin, genistin, malonyl daidzein, malonyl glycitin, malonyl genistin, acetyl daidzein, acetyl glycitin and acetyl genistin. Equal amounts of each standard sample with a concentration of 200 μg / mL were mixed to prepare a mixed standard sample, which was stored at -20°C for use. Qualitative analysis was performed based on the retention time and maximum absorption spectra of the 12 isoflavone standard samples. The ultraviolet absorption value at a wavelength of 260 nm was used as the standard. The content of each isoflavone component and the total isoflavone content in the sample were calculated according to the method described in "Sun J, Sun B, Han F, Yan S, Yang H, Akio K, 2011. Rapid HPLC method for determination of 12 isoflavone components in soybean seeds. Agricultural Sciences in China, 10: 70-77. DOI: 10.1016 / S1671-2927(11)60308-8".

[0094] Compared with the hairy roots containing the empty vector, the daidzein content in the hairy roots overexpressing GmMYB77 (GmMYB77-OE-1, GmMYB77-OE-2, and GmMYB77-OE-3) decreased by 17.0% to 21.5% ( Figure 3b), the content of malonyl daidzein decreased by 19.1% to 21.7% ( Figure 3 c), the content of malonyl glycitin decreased by 15.1% to 24.1% ( Figure 3 d), the total isoflavone content decreased by 26.4% to 29.3% ( Figure 3 e).

[0095] Compared with the hairy roots expressing the empty vector, the daidzein content in the hairy roots silenced by GmMYB77 (GmMYB77-RNAi-1, GmMYB77-RNAi-2, GmMYB77-RNAi-3) increased by 60.6% to 82.5% ( Figure 3 g), the content of malonyl daidzein increased by 28.4% to 34.1% ( Figure 3 h), the content of malonyl glycitin increased by 21.7% to 33.6% ( Figure 3 i), the total isoflavone content increased by 35.7% to 38.9% ( Figure 3 j).

[0096] Therefore, both overexpression and silencing of the GmMYB77 gene significantly changed the isoflavone content in soybean hairy roots, and the GmMYB77 gene negatively regulated the isoflavone content.

[0097] Experimental Example 2

[0098] Transient expression of GmMYB77 gene in tobacco

[0099] 2.1 Construction of GmMYB77 gene overexpression vector

[0100] Using the complete coding region cDNA of GmMYB77 obtained in Experimental Example 1 as a template, PCR was performed using primers pTF101-MYB77-F and pTF101-MYB77-R (Table 1) to amplify the GmMYB77-pTF-OE fragment. The PCR system consisted of 25 μL of 2× Mix, 1 μL of F primer, 1 μL of R primer, 2 μL of template, and 21 μL of sterile water. The PCR program was as follows: first, pre-denaturation at 98°C for 30 seconds; second, denaturation at 98°C for 10 seconds; third, annealing at 57°C for 10 seconds; fourth, extension at 68°C for 1 minute; 30 cycles from steps 2 to 4; and fifth, complete extension at 68°C for 10 minutes.

[0101] Using pTF101-GFP as the starting vector, the GmMYB77 gene overexpression vector pTF101-GmMYB77-GFP ( Figure 4The pTF101-GFP vector is a vector obtained by introducing the GFP open reading frame driven by the CaMV35S promoter and the NOS terminator element into the pTF101.1 vector. The pTF101.1 vector is "Paz MM, Shou H, Guo Z, Zhang ZY, Banerjee AK, Wang K, 2004. Assessment of conditions affecting

[0102] Agrobacterium-mediated soybean transformation using the cotyledonary node explant. Euphytica, 136: 167-179. DOI: 10.1023 / B:EUPH.0000030669.75809.dc”.

[0103] Enzyme digestion reaction system: 5 μL 10× buffer, 15 μL pTF101-GFP plasmid, 2 μL XbaⅠ enzyme and 16 μL sterile water. Reaction procedure: Enzyme digestion at 37℃ for 2 hours. After the enzymatic digestion reaction is completed, check it by 1% agarose gel electrophoresis and use the original plasmid as a control to check whether the plasmid is successfully digested. Use DNA agarose gel recovery kit ( DP209) to recover the linearized pTF101-GFP plasmid. 639649) for ligation reaction: 2 μL 5× In-fusion, 4 μL linearized pTF101-GFP plasmid, 2 μL GmMYB77-pTF-OE fragment and 2 μL sterile water. The ligation product was transformed into Escherichia coli DH5α competent cells, the bacterial solution was spread on LB plates containing spectinomycin, and the plates were inverted and cultured in a 37°C incubator overnight. Single clones were picked and identified by colony PCR using primers GFP-JC-F and GFP-JC-R (Table 1), and the identified positive clones were sequenced. The plasmid of the positive clone with the correct sequence was extracted to obtain the GmMYB77 gene overexpression vector pTF101-GmMYB77-GFP.

[0104] 2.2 Construction of signal carrier

[0105] Plant genomic DNA extraction kit ( Genomic DNA was extracted from leaves of soybean variety "Tianlong No. 1" (TL1) (DP321). PCR was performed using the genomic DNA as a template. Primers PG-IFS1-F and PG-IFS1-R were used to amplify the natural promoter of the isoflavone synthase 1 gene GmIFS1 (Glyma.07G202300) (pGmIFS1), and primers PG-IFS2-F and PG-IFS2-R were used to amplify the natural promoter of the isoflavone synthase 2 gene GmIFS2 (Glyma.13G173500) (pGmIFS1). The native promoter of the gene encoding chalcone synthase 7, GmCHS7 (Glyma.01G228700), was amplified using primers PG-CHS7-F and PG-CHS7-R (pGmCHS7). The native promoter of the gene encoding chalcone synthase 8, GmCHS8 (Glyma.11G011500), was amplified using primers PG-CHS8-F and PG-CHS8-R (pGmCHS8). Glyma.07G202300, Glyma.13G173500, Glyma.01G228700, and Glyma.11G011500 are gene numbers in the soybean genome version Glycine max Wm82.a2.v1. The coding sequences of the genes GmIFS1, GmIFS2, GmCHS7, and GmCHS8 are shown in SEQ ID NOs:12-15. The nucleotide sequences of the promoters pGmIFS1, pGmIFS2, pGmCHS7, and pGmCHS8 are shown in SEQ ID NOs:4-7. The nucleotide sequences of the primers are shown in Table 1. The PCR system consisted of 25 μL 2× Mix, 1 μL F primer, 1 μL R primer, 2 μL template, and 21 μL sterile water. The PCR program was as follows: first step, pre-denaturation at 98°C for 30 s; second step, denaturation at 98°C for 10 s; third step, annealing at 57°C for 10 s; fourth step, extension at 68°C for 1 min; 30 cycles from steps 2 to 4; and fifth step, complete extension at 68°C for 10 min.

[0106] Using the commercially available pGreenII 0800-LUC plasmid (purchased from Beijing Coolbo Technology Co., Ltd., product number: VT307) as the starting vector, a signal vector containing the promoter pGmIFS1 (pGreen-pGmIFS1-LUC), a signal vector containing the promoter pGmIFS2 (pGreen-pGmIFS2-LUC), a signal vector containing the promoter pGmCHS7 (pGreen-pGmCHS7-LUC), and a signal vector containing the promoter pGmCHS8 (pGreen-pGmCHS8-LUC) were constructed. The structure of the signal vector is shown in the figure. Figure 5As shown, wherein promoter represents promoter pGmIFS1, pGmIFS2, pGmCHS7 or pGmCHS8.

[0107] Enzyme digestion reaction system: 5 μL 10× buffer, 15 μL pGreenII 0800-LUC plasmid, 2 μL SmaⅠ enzyme, 2 μL SacⅡ enzyme, and 26 μL sterile water. Reaction procedure: Enzyme digestion at 37°C for 2 h. After the enzymatic digestion reaction is completed, check the plasmid by 1% agarose gel electrophoresis and use the original plasmid as a control to check whether the plasmid is successfully digested. Use DNA agarose gel recovery kit ( DP209) to recover the linearized pGreenII 0800-LUC plasmid. 639649) for a ligation reaction: 2 μL 5× In-fusion, 4 μL linearized pGreenII 0800-LUC plasmid, 2 μL promoter fragment (pGmIFS1, pGmIFS2, pGmCHS7, or pGmCHS8), and 2 μL sterile water. The ligation product was transformed into competent E. coli DH5α cells, plated onto LB plates containing kanamycin, and incubated inverted in a 37°C incubator overnight. Single colonies were picked and identified by colony PCR using primers GmMYB77-colony-PCR-F and GmMYB77-colony-PCR-R (Table 1). Positive clones were sequenced. The plasmids of positive clones with correct sequences were extracted to obtain signal vectors pGreen-pGmIFS1-LUC, pGreen-pGmIFS2-LUC, pGreen-pGmCHS7-LUC and pGreen-pGmCHS8-LUC.

[0108] 2.3 Tobacco transient expression

[0109] Using the heat shock transformation method, pTF101-GFP (control vector) and pTF101-GmMYB77-GFP (effect vector) were introduced into Agrobacterium tumefaciens EHA105 competent cells. Signal vectors pGreen-pGmIFS1-LUC, pGreen-pGmIFS2-LUC, pGreen-pGmCHS7-LUC, and pGreen-pGmCHS8-LUC were introduced into Agrobacterium tumefaciens EHA105 (pSoup) competent cells. The transformation method was as follows: 100 μL of competent cells was added to the plasmid DNA. The mixture was then incubated on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and finally in an ice bath for 5 minutes. Add antibiotic-free LB liquid medium and incubate at 28°C with shaking for 2-3 hours. The cells were collected by centrifugation at 5000 rpm for 1 min, and 100 μL of the supernatant was used to resuspend the cells. The culture was then spread onto LB plates containing the corresponding antibiotics (spectinomycin for pTF101 vector resistance and kanamycin for pGreen vector resistance) and incubated upside down in a 28°C incubator for 2-3 days. Single EHA105 clones were selected and identified by colony PCR using primers GFP-JC-F and GFP-JC-R (Table 1). Positive clones were sequenced to obtain recombinant Agrobacterium EHA105-GFP and EHA105-GmMYB77-GFP. Single clones of EHA105 (pSoup) were picked and identified by colony PCR using primers GmMYB77-colony-PCR-F and GmMYB77-colony-PCR-R (Table 1). The identified positive clones were sequenced to obtain recombinant Agrobacterium EHA105-pGmIFS1-LUC, EHA105-pGmIFS2-LUC, EHA105-pGmCHS7-LUC, and EHA105-pGmCHS8-LUC.

[0110] The above recombinant Agrobacterium single clone was inoculated into YEP liquid medium containing corresponding antibiotics and cultured overnight at 28°C and 200 rpm. 600nm When the value is between 0.6 and 1.0, collect the cells by centrifugation at 1000g for 5 minutes. Gently resuspend the cells in 2 mL of Induction medium (sterile water containing 1% 1M MES and 1% 1M MgCl2), and then collect the cells by centrifugation. Resuspend the cells in 1 mL of Induction medium and let them stand at room temperature for 1 to 4 hours before measuring the OD. 600nm The cells were then collected by centrifugation. The cells were resuspended in infection solution (sterile water containing 1% 1M MES, 1% 1M MgCl2 and 0.01% acetosyringone) and the OD of the bacterial solution was adjusted. 600nmThe infection solution was injected into leaves of Nicotiana benthamiana (Nicotiana benthamiana) grown for 6 to 8 weeks using a syringe without the needle. Four experimental groups and their corresponding control groups were set up as follows.

[0111] In the PIFS1 experimental group, tobacco leaves were injected with a mixture of equal volumes of EHA105-GmMYB77-GFP and EHA105-pGmIFS1-LUC bacterial cultures. In the corresponding control group, tobacco leaves were injected with a mixture of equal volumes of EHA105-GFP and EHA105-pGmIFS1-LUC bacterial cultures.

[0112] In the PIFS2 experimental group, tobacco leaves were injected with a mixture of equal volumes of EHA105-GmMYB77-GFP and EHA105-pGmIFS2-LUC bacterial cultures. In the corresponding control group, tobacco leaves were injected with a mixture of equal volumes of EHA105-GFP and EHA105-pGmIFS2-LUC bacterial cultures.

[0113] PCHS7 experimental group: tobacco leaves were injected with a mixture of equal volumes of EHA105-GmMYB77-GFP and EHA105-pGmCHS7-LUC infection solutions. The corresponding control group was injected with a mixture of equal volumes of EHA105-GFP and EHA105-pGmCHS7-LUC infection solutions.

[0114] PCHS8 experimental group: tobacco leaves were injected with a mixture of equal volumes of EHA105-GmMYB77-GFP and EHA105-pGmCHS8-LUC infection solutions. The corresponding control group was injected with a mixture of equal volumes of EHA105-GFP and EHA105-pGmCHS8-LUC infection solutions.

[0115] After the injection, the tobacco plants were placed in the dark for 12 h and then cultured at 25°C with 16 h of light and 8 h of darkness per day for 48-72 h.

[0116] 2.4 Detection of fluorescence signals in tobacco leaves

[0117] Take tobacco leaves that have been cultured for 48-72 hours and observe whether there is GFP fluorescence signal at the injection site of the leaves using a fluorescent protein observation microscope. Take 0.5g of tobacco leaves with strong GFP signal, grind them with liquid nitrogen, and use a protein rapid extraction kit ( CW0885M) was used to extract cytoplasmic soluble proteins from leaves and placed on ice until ready for use. A Dual-Glo Luciferase Assay System kit (Promega, E1910) stored at -20°C was removed and thawed on ice. The Dual-Glo Luciferase Substrate and Dual-Glo Luciferase Buffer in the kit were mixed (the mixture is referred to as Buffer-Luc) and placed on ice until ready for use. The Dual-Glo Stop & Glo Buffer and Dual-Glo Stop & Glo Substrate were mixed (the mixture is referred to as Buffer-Ren) and placed on ice until ready for use. Tobacco leaf cytoplasmic proteins and Buffer-Luc were added to an ELISA plate and mixed thoroughly by pipetting. The Luc reaction was performed and firefly luciferase activity was measured using a SYNERGY H1 microplate reader. After the assay was complete, 75 μL of Buffer-Ren was added to the mixture and mixed thoroughly by pipetting to terminate the Luc reaction. The Ren reaction was then initiated and Renilla luciferase activity was measured. Using Renilla luciferase (CaMV 35S::Ren) as an internal reference, the RLU (relative light unit) value measured after the Luc reaction was divided by the RLU value measured after the Ren reaction to calculate the LUC / REN ratio. The regulatory effect of the transcription factor GmMYB77 on the native promoters of the GmIFS1, GmIFS2, GmCHS7, and GmCHS8 genes was analyzed by comparing the LUC / REN ratios of the experimental and control groups.

[0118] Compared with the control group tobacco leaves without GmMYB77 expression, the promoter activity of the GmIFS1 gene (pGmIFS1) in the experimental group tobacco leaves expressing GmMYB77 decreased by 62.8% ( Figure 6 b), the promoter activity of GmIFS2 gene promoter (pGmIFS2) decreased by 62.1% ( Figure 6 c), the promoter activity of GmCHS7 gene promoter (pGmCHS7) decreased by 87.2% ( Figure 6 d), the promoter activity of GmCHS8 gene promoter (pGmCHS8) decreased by 85.9% ( Figure 6 e). Therefore, GmMYB77 significantly inhibited the expression of genes encoding key enzymes in the isoflavone biosynthesis pathway (isoflavone synthase 1, isoflavone synthase 2, chalcone synthase 7, and chalcone synthase 8). This suggests that GmMYB77 regulates the isoflavone content in soybeans by inhibiting the expression of genes encoding key enzymes in the isoflavone biosynthesis pathway.

[0119] Experimental Example 3

[0120] Overexpression and knockout of GmMYB77 gene in soybean plants

[0121] 3.1 Vector construction

[0122] Based on the sequence characteristics of the GmMYB77 gene, a suitable sgRNA target sequence (SEQ ID NO: 9) was designed and screened online using CRISPR-P2.0. The U6 promoter (SEQ ID NO: 8), the gRNA targeting GmMYB77 (SEQ ID NO: 9), and the gRNA scaffold (SEQ ID NO: 10) were sequentially connected using PCR to generate the U6-gRNA fragment (SEQ ID NO: 11).

[0123] Using the empty pU3-gRNA vector as a template, PCR was performed using primers U6-F and U6-R (Table 1) to amplify the U6 fragment. The PCR system consisted of 25 μL 2× Mix, 1 μL F primer, 1 μL R primer, 2 μL template, and 21 μL sterile water. The PCR program was as follows: first, pre-denaturation at 98°C for 30 s; second, denaturation at 98°C for 10 s; third, annealing at 57°C for 10 s; fourth, extension at 68°C for 1 min; 30 cycles from steps 2 to 4; and fifth, complete extension at 68°C for 10 min. Using the empty pU3-gRNA vector as a template, PCR was performed using primers GmMYB77-gRNA-F and RPS5A-R (Table 1) to amplify the gRNA-gRNA scaffold fragment. The PCR system consisted of 25 μL 2× Mix, 1 μL F primer, 1 μL R primer, 2 μL template, and 21 μL sterile water. The PCR program was as follows: first, pre-denaturation at 98°C for 30 seconds; second, denaturation at 98°C for 10 seconds; third, annealing at 57°C for 10 seconds; fourth, extension at 68°C for 1 minute; 30 cycles from steps 2 to 4; and fifth, complete extension at 68°C for 10 minutes. PCR was performed using the U6 fragment (template 1) and the gRNA-gRNA scaffold fragment (template 2) as templates using primers RPS5A-F and RPS5A-R (Table 1) to amplify the U6-gRNA fragment. The PCR system consisted of 25 μL 2× Mix, 1 μL F primer, 1 μL R primer, 2 μL template 1, 2 μL template 2, and 19 μL sterile water. The PCR program was as follows: first, pre-denaturation at 98°C for 30 seconds; second, denaturation at 98°C for 10 seconds; third, annealing at 57°C for 10 seconds; fourth, extension at 68°C for 1 minute; 30 cycles from steps 2 to 4; and fifth, complete extension at 68°C for 10 minutes.

[0124] Using RPS5A::Cas9 vector as the starting vector, the above U6-gRNA fragment was inserted into the XbaI restriction site of the vector to obtain the GmMYB77 gene editing vector GmMYB77-CRISPR / Cas9 ( Figure 7 The RPS5A::Cas9 vector is the CRISPR / Cas9 expression vector described in “Li C, LiYH, Li Y, Lu H, Hong H, Tian Y, Li H, Zhao T, Zhou X, Liu J, Zhou X, Jackson SA, Liu B, Qiu LJ, 2020. A domestication-associated gene GmPRR3b regulates the circadian clock and flowering time in soybean. Molecular Plant. 2020. DOI: 10.1016 / j.molp.2020.01.014”.

[0125] Enzyme digestion reaction system: 5 μL 10× buffer, 15 μL RPS5A::Cas9 plasmid, 2 μL XbaI enzyme and 28 μL sterile water. Reaction procedure: Enzyme digestion at 37℃ for 2 hours. After the enzymatic digestion reaction is completed, it is detected by 1% agarose gel electrophoresis and the original plasmid is used as a control to check whether the plasmid is successfully digested. Use DNA agarose gel recovery kit ( DP209) to recover the linearized RPS5A::Cas9 plasmid. 639649) for ligation reaction: 2 μL 5× In-fusion, 4 μL linearized RPS5A::Cas9 plasmid, 2 μL U6-gRNA fragment and 2 μL sterile water. The ligation product was transformed into Escherichia coli DH5α competent cells, and the bacterial solution was spread on LB plates containing kanamycin and inverted in a 37°C incubator for overnight culture. Single clones were picked and identified by colony PCR using primers RPS5A-F and RPS5A-R (Table 1), and the identified positive clones were sequenced. The plasmid of the positive clone with the correct sequence was extracted to obtain the GmMYB77 gene editing vector GmMYB77-CRISPR / Cas9.

[0126] 3.2 Soybean genetic transformation

[0127] Following the transformation method described in Experimental Example 2 (2.3), the GmMYB77 gene-editing vector GmMYB77-CRISPR / Cas9 was introduced into Agrobacterium tumefaciens EHA105 competent cells. Single colonies were selected using kanamycin, and colony PCR was performed using primers RPS5A-F and RPS5A-R (Table 1). Positive clones were sequenced to obtain recombinant Agrobacterium tumefaciens EHA105-GmMYB77-CRISPR / Cas9.

[0128] Soybean genetic transformation was performed using the recombinant Agrobacterium EHA105-GmMYB77-GFP containing the GmMYB77 gene overexpression vector pTF101-GmMYB77-GFP described in Experimental Example 2 and the aforementioned recombinant Agrobacterium EHA105-GmMYB77-CRISPR / Cas9, respectively, to obtain GmMYB77-overexpressing T0 transgenic plants and GmMYB77-knockout T0 transgenic plants. The transformation method is as follows.

[0129] Seed sterilization: Soybean variety "Tianlong No. 1" (TL1) seeds were placed in a culture dish and sterilized using chlorine sterilization. After sterilization, the seeds were placed on a clean bench and blown for 15 minutes to remove residual chlorine.

[0130] Seed germination: Take the sterilized soybean seeds, plant them with the hilum facing down in the germination medium, seal them and place them in a culture room at 25°C, with 16 hours of light and 8 hours of darkness per day, and culture them for 16 hours.

[0131] Preparation of bacterial solution: Take 40-60 μL of Agrobacterium bacterial solution and evenly spread it on LB solid medium containing corresponding antibiotics, and incubate it upside down in a dark incubator at 28°C for 48 hours; after activation, resuspend it with double distilled water, and then evenly spread it on LB solid medium containing corresponding antibiotics, seal it and invert it in a dark incubator at 28°C for 24 hours, then scrape the bacteria and resuspend them in liquid induction medium to the bacterial solution OD 600nm The value is 0.6-0.8, and the infected bacterial solution is obtained.

[0132] Preparation of cotyledonary node explants: remove the seed coat, separate the two cotyledons, gently remove the cotyledons with a knife, and gently cut 3-5 times at the connection between the cotyledonary nodes to obtain the cotyledonary node explants.

[0133] Infection: Soak the cotyledonary node explants in the infection solution for 1.5 hours.

[0134] Co-cultivation: After infection, place the cotyledonary node explants with the convex side facing down on CCM medium covered with sterile filter paper, seal the culture dish and culture in the dark at 22°C for 5 days.

[0135] Resume induction culture: Remove the cotyledonary node explants after 5 days of co-cultivation. Rinse the explants 4-5 times with liquid induction medium without sucrose or agar. Blot dry the surface with filter paper and insert them into solid induction medium at a 45° angle. Seal the culture dish and place it in a culture room at 25°C with 16 hours of light and 8 hours of darkness per day for 7 days.

[0136] Screening and Induction Culture: After resuming induction culture, remove the excessively long hypocotyls and clustered buds, cut off part of the radicle, and retain approximately 0.5 cm of the radicle. Insert the explant into the screening medium at a 45° angle. Seal the explant and place it in a culture room at 25°C with 16 hours of light and 8 hours of darkness per day for 21 days.

[0137] Elongation subculture: Take the explants that have been screened and induced for 21 days, cut off part of the hypocotyl, and insert them into the elongation medium at a 45° angle. Culture them in a sealed container at 25°C, with 16 hours of light and 8 hours of darkness per day. The first elongation culture is carried out for 21 days, followed by elongation subcultures for 15 days each. Repeat the subculture process 2-3 times.

[0138] Rooting culture: Transfer 8-10 cm transformed seedlings from elongation medium to rooting medium. Incubate in a sealed container at 25°C with 16 hours of light and 8 hours of darkness daily. When the transformed seedlings develop approximately 10 roots, transplant them to nutrient soil for cultivation.

[0139] The formula of the above-mentioned germination medium is as follows: 3.21 g / L B5 medium, 20 g / L sucrose, adjust the pH to 5.8 with NaOH (1 M), add 7 g / L agar, sterilize at 120°C for 20 min, wait until the medium temperature drops to 50°C, add 1 mL / L B5 organic salt to the medium, mix well, and pour into a disposable plastic dish.

[0140] The formula of the above-mentioned CCM medium is: 0.32g / L B5 medium, 30g / L sucrose, 3.9g / L MES, add 900mL ultrapure water, adjust the pH to 5.4 with NaOH (1M), add ultrapure water to 1L, add 5g / L agar, sterilize at 120℃ for 20min, wait until the culture medium temperature drops to 50℃, add 1mL / L B5 vitamin, 1mL / L AS (40mg / mL), 1mL / LDTT (154mg / mL), 4mL / L Na2S2O3 (158mg / mL), mix well and pour into a disposable culture dish.

[0141] The formula of the above-mentioned liquid induction medium is as follows: 3.2 g / L B5 medium, 30 g / L sucrose, 0.6 g / L MES, add ultrapure water to 1 L, adjust the pH to 5.7 with NaOH (1 M), sterilize at 120°C for 20 min, wait until the culture medium temperature drops to 50°C, add 1 mL / L B5 vitamins, 1 mL / L PPT (5 mg / mL), 1 mL / L vancomycin (50 mg / mL), 1 mL / L cephalosporin (50 mg / mL), and 1 mL / L timentin (100 mg / mL), mix well, and pour into a disposable culture dish.

[0142] The formula of the above-mentioned solid induction medium is as follows: 3.2 g / L B5 medium, 30 g / L sucrose, 0.6 g / L MES, add ultrapure water to 1 L, adjust the pH to 5.7 with NaOH (1 M), add 7 g / L agar, sterilize at 120°C for 20 min, wait until the culture medium temperature drops to 50°C, add 1 mL / L B5 vitamins, 1 mL / L vancomycin (50 mg / mL), 1 mL / L cephalosporin (50 mg / mL), and 1 mL / L timentin (100 mg / mL), mix well, and pour into a disposable culture dish.

[0143] The formula of the above screening medium is: 3.2g / L B5 medium, 30g / L sucrose, 0.6g / L MES, add ultrapure water to 1L, adjust the pH to 5.7 with NaOH (1M), add 7g / L agar, sterilize at 120℃ for 20min, wait until the culture medium temperature drops to 50℃, add 1mL / L B5 vitamin, 1mL / L PPT (5mg / mL), 1mL / L vancomycin (50mg / mL), 1mL / L cephalosporin (50mg / mL), 1mL / L timentin (100mg / mL), mix well and pour into a disposable culture dish.

[0144] The formula of the above-mentioned elongation medium is as follows: 4.3 g / L MSB medium, 30 g / L sucrose, 0.6 g / L MES, add ultrapure water to 1 L, adjust the pH to 5.7 with NaOH (1 M), add 7 g / L agar, sterilize at 120°C for 20 min, wait until the culture medium temperature drops to 50°C, add 1 mL / L B5 vitamin, 1 mL / L PPT (5 mg / mL), 1 mL / L vancomycin (50 mg / mL), 1 mL / L cephalosporin (50 mg / mL), 1 mL / L timentin (100 mg / mL), 0.1 mL / LIAA (1 mg / mL), 0.2 mL / L zeatin (5 mg / mL), 1 mL / L gibberellin (1 mg / mL) to the culture medium, mix well and pour into a disposable culture dish.

[0145] The formula of the above-mentioned rooting medium is as follows: 2.2 g / L MSB medium, 20 g / L sucrose, 0.6 g / L MES, add ultrapure water to 1 L, adjust the pH to 5.7 with NaOH (1 M), add 7 g / L agar, sterilize at 120°C for 20 min, wait until the culture medium temperature drops to 50°C, add 1 mL / L B5 vitamin, 1 mL / L vancomycin (50 mg / mL), 1 mL / L cephalosporin (50 mg / mL), 1 mL / L timentin (100 mg / mL), 1 mL / L indolebutyric acid (1 mg / mL) to the culture medium, mix well and pour into a disposable culture dish.

[0146] 3.3 Screening of GmMYB77 Overexpressing Plants

[0147] Bar gene testing was performed on T0 transgenic plants overexpressing GmMYB77 to screen for transgenic-positive plants. The method is as follows: a small amount of soybean leaves were placed in a 1.5 mL centrifuge tube. A small amount of water was added to the leaf tissue to break it up. A BAR rapid test strip was inserted into the broken leaf tissue. After waiting for approximately 3 minutes, the test results were observed when a band appeared on the control line of the test strip. If a band appeared on the test line, the plant was transgenic-positive.

[0148] RNA was extracted from leaves of transgenic positive plants and reverse transcribed into cDNA. The expression level of GmMYB77 gene was detected according to the real-time fluorescence quantitative PCR method in Experimental Example 1 (1.5).

[0149] Proteins were extracted from leaves of transgenic plants, separated by SDS-PAGE, and blotted onto PVDF membranes. GFP protein was detected by Western blotting using Anti-GFP Mouse Monoclonal Antibody (Beijing Quanshijin Biotechnology Co., Ltd., HT801-01) and Goat Anti-Mouse IgG (H+L), HRP Conjugate (Beijing Quanshijin Biotechnology Co., Ltd., HS201-01) as secondary antibodies.

[0150] Test results such as Figure 8 A total of three homozygous GmMYB77 overexpression lines were screened: GmMYB77-1, GmMYB77-2, and GmMYB77-4.

[0151] 3.4 Phenotypic Analysis of GmMYB77 Overexpressing Plants

[0152] Seeds of the stable inherited T4 generation homozygous GmMYB77 overexpression lines (GmMYB77-1, GmMYB77-2, and GmMYB77-4) were selected and the isoflavone content was determined according to the method in Experimental Example 1 (1.6).

[0153] The results showed that compared with the "Tianlong No. 1" soybean (TL1), the malonyl glycitin content in the seeds of the three GmMYB77 overexpression lines (GmMYB77-1, GmMYB77-2 and GmMYB77-4) decreased by 24.1% to 30.4% ( Figure 9 a), the content of malonyl genistin decreased by 9.2% to 14.1% ( Figure 9 b), the total isoflavone content decreased by 13.7% to 22.8% ( Figure 9 c).

[0154] RNA was extracted from leaves of GmMYB77-overexpressing lines (GmMYB77-1, GmMYB77-2, and GmMYB77-4) and reverse-transcribed into cDNA. Real-time quantitative PCR was performed using TaKaRa's SYBR Premix Ex Taq II using cDNA as template to measure the relative expression levels of GmIFS1 (SEQ ID NO: 12), GmIFS2 (SEQ ID NO: 13), GmCHS7 (SEQ ID NO: 14), GmCHS8 (SEQ ID NO: 15), and GmIOMT1 (SEQ ID NO: 16), key enzymes in the isoflavone biosynthesis pathway. The internal reference gene was GmActin-6 (SEQ ID NO: 3). Primers used for amplifying GmIFS1 were qPCR-GmIFS1-F and qPCR-GmIFS1-R. Primers used for amplifying GmIFS2 were qPCR-GmIFS2-F and qPCR-GmIFS2-R. The primers used to amplify GmCHS7 are qPCR-GmCHS7-F and qPCR-GmCHS7-R. The primers used to amplify GmCHS8 are qPCR-GmCHS8-F and qPCR-GmCHS8-R. The primers used to amplify GmIOMT1 are qPCR-GmIOMT1-F and qPCR-GmIOMT1-R. The nucleotide sequences of the primers are shown in Table 1. The PCR system is: 5 μL 2×Mix, 0.4 μL F primer, 0.4 μL R primer, 1 μL cDNA and 3.2 μL sterile water. The PCR program is: the first step is pre-denaturation at 98°C for 3 minutes; the second step is denaturation at 98°C for 10 seconds; the third step is annealing at 57°C for 1 minute; the fourth step is extension at 72°C for 1 minute; 30 cycles are set from the second to the fourth step; the fifth step is complete extension at 72°C for 3 minutes. The data were analyzed using Excel software and 2 -ΔΔCTThe relative expression level of each gene was calculated.

[0155] The results showed that the expression of GmIFS1 gene in leaves of three GmMYB77 overexpression lines (GmMYB77-1, GmMYB77-2 and GmMYB77-4) decreased by 38.5% to 68.5% compared with "Tianlong No. 1" soybean (TL1). Figure 9 d), GmIFS2 gene expression decreased by 84.2% to 87.2% ( Figure 9 e), the expression of GmCHS7 gene decreased by 74.5% to 83.9% ( Figure 9 f), the expression level of GmCHS8 gene decreased by 94.0% to 95.5% ( Figure 9 g), GmIOMT1 gene expression decreased by 40.3% to 55.3% ( Figure 9 h).

[0156] The above results showed that overexpression of GmMYB77 could inhibit the expression of GmIFS1, GmIFS2, GmCHS7, GmCHS8 and GmIOMT1 genes in the soybean isoflavone biosynthesis pathway and reduce the isoflavone content, among which the content of malonyl glycitin decreased the most.

[0157] Screening of 3.5GmMYB77 knockout plants

[0158] According to the Bar gene detection method in 3.3 above, the GmMYB77 knockout T0 generation transgenic plants were tested for Bar gene to screen transgenic positive plants.

[0159] Transgenic positive plants were sown individually and T1 transgenic plants were harvested. Plant genomic DNA extraction kit ( Genomic DNA was extracted from leaves of T1 transgenic plants at the flowering stage (DP321). PCR was performed using genomic DNA as a template, using primers Bar-F and Bar-R to amplify the Bar gene, and primers CR-GmMYB77-F and CR-GmMYB77-R to amplify the editing target gene. The amplified products were sequenced. The nucleotide sequences of the primers are shown in Table 1. The PCR system consisted of 5 μL 2× Mix, 0.4 μL F primer, 0.4 μL R primer, 1 μL genomic DNA, and 3.2 μL sterile water. The PCR program was as follows: first, pre-denaturation at 98°C for 3 minutes; second, denaturation at 98°C for 10 seconds; third, annealing at 57°C for 1 minute; fourth, extension at 72°C for 1 minute; 30 cycles from steps 2 to 4; and fifth, complete extension at 72°C for 3 minutes.

[0160] Three GmMYB77 homozygous knockout mutants were screened from the T1 transgenic plants: Gmmyb77-1, Gmmyb77-2, and Gmmyb77-3. All of them were deletion mutants, with deletions of 1 bp, 4 bp, and 81 bp, respectively. Figure 10 a).

[0161] The T1 generation GmMYB77 homozygous knockout plants were sown individually and the T2 generation GmMYB77 homozygous knockout plants were harvested. RNA was extracted from the leaves of the T2 generation GmMYB77 homozygous knockout plants and reverse transcribed into cDNA. The expression level of the GmMYB77 gene in the leaves was detected according to the real-time fluorescence quantitative PCR method in Experimental Example 1 (1.5). Figure 10 As shown in b, compared with "Tianlong No. 1" soybean (TL1), the expression levels of GmMYB77 gene in three GmMYB77 homozygous knockout materials (Gmmyb77-1, Gmmyb77-2 and Gmmyb77-3) were reduced by 90.5%, 85.2% and 85.1%, respectively.

[0162] Genomic DNA was extracted from leaves of T2 generation GmMYB77 homozygous knockout plants, and the Bar gene was amplified using primers Bar-F and Bar-R (Table 1). At the same time, the Bar gene expression in leaves of T2 generation GmMYB77 homozygous knockout plants was detected using BAR rapid test strips. Figure 10 As shown in Figures c and 10d, the Bar gene could not be detected in the three GmMYB77 homozygous knockout materials (Gmmyb77-1, Gmmyb77-2, and Gmmyb77-3), indicating that the CRISPR / Cas9 gene-edited plants were Bar gene-detransfected plants.

[0163] 3.6 Phenotypic analysis of GmMYB77 knockout plants

[0164] T2 generation GmMYB77 homozygous knockout plants were sown individually and T3 generation GmMYB77 homozygous knockout plants were harvested. The isoflavone content in the seeds of the T3 generation GmMYB77 homozygous knockout plants was determined according to the method in Experimental Example 1 (1.6).

[0165] The results showed that compared with the "Tianlong No. 1" soybean (TL1), the malonyl glycitin content in the seeds of three GmMYB77 homozygous knockout materials (Gmmyb77-1, Gmmyb77-2 and Gmmyb77-3) was significantly increased by 28.1% to 58.7% ( Figure 11 a), the content of malonyl genistin increased significantly by 12.2% to 16.0% ( Figure 11 b), the total isoflavone content increased significantly by 11.0% to 15.2% ( Figure 11 c).

[0166] RNA was extracted from leaves of T3 Gmmyb77 homozygous knockout plants and reverse transcribed into cDNA. The relative expression levels of the key enzyme genes in the isoflavone biosynthesis pathway, GmIFS1 (SEQ ID NO: 12), GmIFS2 (SEQ ID NO: 13), GmCHS7 (SEQ ID NO: 14), GmCHS8 (SEQ ID NO: 15), and GmIOMT1 (SEQ ID NO: 16), were determined using the real-time fluorescence quantitative PCR method described in 3.4 above.

[0167] The results showed that the expression level of GmIFS1 gene in leaves of three GmMYB77 homozygous knockout materials (Gmmyb77-1, Gmmyb77-2 and Gmmyb77-3) was significantly increased by 43.8% to 70.3% compared with "Tianlong No. 1" soybean (TL1). Figure 11 d), the expression of GmIFS2 gene increased significantly by 1.53-4.12 times ( Figure 11 e), the expression of GmCHS7 gene increased significantly by 5.55-9.22 times ( Figure 11 f), the expression level of GmCHS8 gene increased significantly by 5.59-10.54 times ( Figure 11 g), the expression of GmIOMT1 gene increased significantly by 374.20-519.36 times ( Figure 11 h).

[0168] The above results showed that knocking out the GmMYB77 gene could increase the expression levels of GmIFS1, GmIFS2, GmCHS7, GmCHS8 and GmIOMT1 genes in the isoflavone synthesis pathway, thereby increasing the isoflavone content, among which the content of malonyl glycitin increased the most.

Claims

1. A method for regulating the isoflavone content in plants, comprising: The level of transcription factor GmMYB77 in a plant is reduced to increase the isoflavone content in the plant, or the level of transcription factor GmMYB77 in a plant is increased to reduce the isoflavone content in the plant; the amino acid sequence of the transcription factor GmMYB77 is shown in SEQ ID NO: 2; and the plant is soybean.

2. The method of claim 1, wherein the level of the transcription factor GmMYB77 in the plant is reduced by silencing or knocking out the gene encoding the transcription factor GmMYB77 in the plant, or the level of the transcription factor GmMYB77 in the plant is increased by overexpressing the gene encoding the transcription factor GmMYB77 in the plant.

3. The method of claim 2, wherein the nucleotide sequence of the gene encoding the transcription factor GmMYB77 is shown in SEQ ID NO:

1.

4. The method of claim 3, wherein the gene encoding the transcription factor GmMYB77 is knocked out in the plant using the CRISPR / Cas9 system.

5. The method of claim 4, wherein the target sequence of the gene encoding the transcription factor GmMYB77 used in the CRISPR / Cas9 system is shown in SEQ ID NO:

9.

6. The method according to claim 5, wherein the gene encoding the transcription factor GmMYB77 in the plant is knocked out by introducing a gene encoding an sgRNA targeting the target sequence and a gene encoding Cas9 into the plant.

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