Application of soybean C2H2 type zinc finger protein transcription factor GmZFP7 and / or its gene in regulating isoflavones

Abstract

The application of soybean C2H2 type zinc finger protein transcription factor GmZFP7 and / or its gene in regulating isoflavones belongs to the field of plant genetic engineering.The application provides the application of soybean C2H2 type zinc finger protein transcription factor GmZFP7 in regulating isoflavones, the application of gene GmZFP7 with gene accession number Gene ID:100792169 in regulating isoflavones, and further provides an isoflavone regulating method.The application proves by a large number of experiments that the total isoflavone content in the transgenic plant overexpressing the transcription factor gene GmZFP7 is significantly increased, and the total isoflavone content in the mutant plant knocking out the gene GmZFP7 by gene editing is significantly reduced.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically involving the application of soybean C2H2 type zinc finger protein transcription factor GmZFP7 and / or its gene in regulating isoflavones. Background Technology

[0002] Isoflavones are important secondary metabolites synthesized in plants, especially legumes. They play crucial roles in both plants and animals. In animals, particularly humans, their chemical structure is similar to estrogen and they possess estrogen-like activity, playing important roles in anti-cancer activity, improving osteoporosis, reducing cardiovascular disease, and preventing and treating menopausal syndrome in women. In plants, isoflavones play a vital role in resisting pathogen infection, inducing soybean nodulation as signaling molecules, and ensuring normal plant growth and development. Due to the significant application value of isoflavones in food and health products, they have received widespread attention. Soybeans are a major natural source of isoflavones; therefore, developing high-isoflavone specialty soybean varieties is one of the main goals of soybean nutritional quality breeding.

[0003] Genetic engineering is an effective means of improving crop traits. Currently, increasing soybean isoflavone content mainly involves modifying the structural genes of isoflavone synthases and key enzyme genes in the isoflavone competitive pathway. However, transgenic plants often suffer from survival difficulties due to the blockage of metabolic pathways for important secondary metabolites such as flavonoids and anthocyanins. Furthermore, some MYB-type transcription factors have been identified that influence soybean isoflavone accumulation by regulating the expression levels of key enzyme genes in the isoflavone synthesis pathway. However, the number of identified transcription factor genes regulating isoflavone content remains small, and in-depth functional mechanism studies are lacking.

[0004] GmZFP7 (Glyma.20G012700, Gene ID: 100792169) is a gene encoding soybean zinc finger protein 7, which encodes the soybean C2H2 type zinc finger protein transcription factor GmZFP7. Functional identification of this gene and protein has not been reported in this field, nor has the relationship between this gene, protein, and isoflavone content been reported before. Summary of the Invention

[0005] Based on the aforementioned gaps in the prior art, this invention provides the application of the soybean C2H2 type zinc finger protein transcription factor GmZFP7, whose amino acid sequence is shown in SEQ ID NO.2, or the gene encoding it with Gene ID: 100792169, in regulating isoflavones.

[0006] The technical solution of the present invention is as follows:

[0007] Application of soybean C2H2 type zinc finger protein transcription factor GmZFP7, whose amino acid sequence is shown in SEQ ID NO.2, in regulating isoflavones.

[0008] The gene accession number for the soybean C2H2 type zinc finger protein transcription factor GmZFP7 is Gene ID: 100792169.

[0009] The nucleotide sequence of the gene GmZFP7, which is the soybean C2H2 type zinc finger protein transcription factor GmZFP7, is shown in SEQ ID NO.1;

[0010] The regulation of isoflavones refers to increasing or decreasing the level of soybean C2H2 type zinc finger protein transcription factor GmZFP7 in plants, or increasing or decreasing the isoflavone content in plants by overexpressing, silencing, knocking down, or knocking out the gene GmZFP7 of soybean C2H2 type zinc finger protein transcription factor GmZFP7 in plants.

[0011] The plants were selected from soybeans and tobacco.

[0012] Application of the gene GmZFP7 with gene accession number Gene ID:100792169 in the regulation of isoflavones.

[0013] The gene locus number of GmZFP7 is Glyma.20G012700; the nucleotide sequence of the gene GmZFP7 is shown in SEQ ID NO.1.

[0014] The gene GmZFP7 regulates isoflavones by activating the expression of the isoflavone synthase 2 gene GmIFS2 and / or inhibiting the expression of the flavanone-3-hydroxylase 1 gene GmF3H1.

[0015] The regulation of isoflavones refers to increasing or decreasing the isoflavone content in plants.

[0016] Preferably, the plant is selected from soybeans and tobacco.

[0017] A method for regulating isoflavones, characterized in that isoflavones are regulated by controlling the level of soybean C2H2 type zinc finger protein transcription factor GmZFP7 with amino acid sequence as shown in SEQ ID NO.2, and / or by controlling the expression of gene GmZFP7 with gene accession number Gene ID:100792169.

[0018] The level of soybean C2H2 zinc finger protein transcription factor GmZFP7 (as shown in SEQ ID NO.2) can be regulated by overexpressing, silencing, knocking down, or knocking out the gene GmZFP7, and / or by regulating the expression of the gene GmZFP7 with gene accession number 100792169.

[0019] The overexpression refers to: plants transformed with the recombinant overexpression vector PTF101-GmZFP7-GFP or pGGP-GmZFP7 obtained by ligating the gene GmZFP7 into the expression vector PTF101-GFP or pGGP.

[0020] Preferably, the primer sequences for ligating the gene GmZFP7 into the expression vector are shown in SEQ ID NO.5-6;

[0021] The silencing expression refers to the recombinant silencing expression vector pGGP-GmZFP7-RNAi transformed into plants by ligating the RNAi sequence of the gene GmZFP7 into the vector pGGP;

[0022] Preferably, the primer sequences for ligating the RNAi sequence of the gene GmZFP7 into the vector pGGP are shown in SEQ ID NO.7-10;

[0023] The knockout refers to: transforming plants with the JRH0645-GmZFP7 gene editing vector;

[0024] Preferably, the JRH0645-GmZFP7 gene editing vector is constructed by using JRH0645 (CaMV35s:Cas9) as the original vector and inserting the U6 promoter, the gRNA targeting GmZFP7 and the gRNA scaffold in tandem into the XbaI restriction site using PCR.

[0025] Preferably, the target sequence of the gRNA is shown in SEQ ID NO.31.

[0026] This invention discloses the application of a soybean C2H2 type zinc finger protein transcription factor GmZFP7 and its encoding gene GmZFP7 (gene locus number: Glyma.20G012700) in regulating soybean isoflavone content. The GmZFP7 transcription factor has the amino acid sequence shown in SEQ ID No. 2. The open reading frame of this gene has the DNA sequence shown in SEQ ID No. 1. The transcription factor GmZFP7 of this invention has bifunctional transcription factor activity, capable of activating the expression of isoflavone synthase 2 (IFS2), a key enzyme in the soybean isoflavone synthesis pathway, while simultaneously inhibiting the expression of flavanone 3-hydroxylase 1 (F3H1), a key enzyme in the flavonol synthesis pathway.

[0027] Overexpression of the open reading frame (OPF) of the coding gene for this transcription factor in soybean hair roots significantly increased the total isoflavone content; conversely, inhibiting the expression of the coding gene significantly decreased the total isoflavone content. In stable transgenic soybean plants, the total isoflavone content in leaves and seeds of transgenic plants overexpressing this transcription factor gene was significantly increased, while the total isoflavone content in leaves and seeds of mutant plants with the gene knocked out by gene editing was significantly decreased. This invention has significant application value for breeding new soybean varieties with different levels of isoflavone content. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the structure of the gene overexpression vector pGGP-GmZFP7 based on GFP detection, which is the experimental method section of the first part of the experimental examples of this invention, specifically in the experimental methods section 3.

[0029] Figure 2 This is a schematic diagram of the structure of the gene silencing vector pGGP-GmZFP7-RNAi based on GFP detection, which is the experimental method section of the first part of the experimental examples of this invention.

[0030] Figure 3 This is a schematic diagram of the structure of the plant expression vector PTF101-GmZFP7-GFP, which is described in Section 3, Experimental Methods, of the first part of the Experimental Examples of this invention.

[0031] Figure 4 This is a schematic diagram of the structure of the plant expression vector pGreen-promoter-LUC in Section 3 of the Experimental Methods section of the first part of the Experimental Examples of this invention.

[0032] Figure 5 This is a diagram of the JRH0645-GmZFP7 gene editing vector from Section 3, Experimental Methods, Part 1 of the Experimental Examples of this invention.

[0033] Figure 6 This is a bar graph showing the isoflavone content and relative expression level of GmZFP7 in LHD hair roots, as described in Section 1 of the experimental results of Part 2 of the experimental examples of this invention; where (A) represents the relative expression level of GmZFP7 in hair roots, and (B) represents the relative content of total isoflavones in hair roots with GmZFP7 overexpression and silence.

[0034] Figure 7 The diagram shows the vector and bar graph of the induction regulation of GmZFP7 on the promoter activities of GmIFS2 and GmF3H1 in Section 2 of the experimental results of Part 2 of the experimental examples of this invention; where (A) is a schematic diagram of the tobacco transient expression vector; and (B) is a bar graph of the induction level of GmZFP7 on the promoter activities of GmIFS2 and GmF3H1.

[0035] Figure 8 The results of obtaining and detecting the GmZFP7 overexpression transgenic lines are presented in Section 3.1 of the experimental results of Part 2 of the present invention. (A) shows the GmZFP7 overexpression lines and control lines; (B) shows the BAR test strip detection results of the GmZFP7 overexpression plants. The figure shows that the three GmZFP7 overexpression lines GmZFP7-OE1, GmZFP7-OE2 and GmZFP7-OE3 all have positive bands for herbicide resistance genes; (C) shows the resistance effect of transgenic leaves to glufosinate. The leaves with positive results are from the GmZFP7 overexpression line GmZFP7-OE2. The leaves of GmZFP7-OE2 and GmZFP7-OE3 have similar phenotypes and will not be repeated.

[0036] Figure 9 The results of phenotypic identification of the GmZFP7 overexpression transgenic lines are presented in Section 3.2 of the experimental results of Part II of the present invention; (A) is a bar chart of GmZFP7 gene expression level in leaves of the overexpression lines; (B) is a bar chart of isoflavone content changes in leaves of the GmZFP7 overexpression lines; (C) is a bar chart of isoflavone content changes in seeds of the GmZFP7 overexpression lines; and (D) is a bar chart of GmIFS2 and GmF3H1 expression levels changes in leaves of the overexpression lines.

[0037] Figure 10 This is the screening process and results of CRISPR / Cas9-mediated GmZFP7 knockout plants in Section 3.3 of the experimental results of Part 2 of the present invention; (A) shows the location and sequence of gRNA in GmZFP7; (B) shows the PCR detection electrophoresis diagram of the CRISPR / Cas9 sequence; (C) shows the Williams82 control and GmZFP7 knockout mutant seedlings; (D) shows the screening of GmZFP7 knockout mutants.

[0038] Figure 11 The results are as follows: (A) is a bar chart showing the total isoflavone content in the leaves of the Gmzfp7 mutant; (B) is a bar chart showing the total isoflavone content in the seeds of the Gmzfp7 mutant; (C) is a bar chart showing the change in the expression level of GmZFP7 in the leaves of the Gmzfp7 mutant; and (D) is a bar chart showing the change in the expression levels of GmIFS2 and GmF3H1 in the leaves of the Gmzfp7 mutant. Detailed Implementation

[0039] The following detailed description of the present invention, in conjunction with specific embodiments and experimental examples, does not limit the scope of protection of the present invention.

[0040] Group 1 Examples: New Applications of the Transcription Factor GmZFP7 of the Present Invention

[0041] This set of examples provides the application of soybean C2H2 type zinc finger protein transcription factor GmZFP7, with an amino acid sequence as shown in SEQ ID NO.2, in regulating isoflavones.

[0042] In a specific embodiment, the gene accession number of the gene encoding the soybean C2H2 type zinc finger protein transcription factor GmZFP7 is Gene ID: 100792169.

[0043] In a more specific embodiment, the nucleotide sequence of the gene GmZFP7 of the soybean C2H2 type zinc finger protein transcription factor GmZFP7 is shown in SEQ ID NO.1;

[0044] Preferably, the regulation of isoflavones refers to increasing or decreasing the level of soybean C2H2 type zinc finger protein transcription factor GmZFP7 in the plant, or increasing or decreasing the isoflavone content in the plant by overexpressing, silencing, knocking down, or knocking out the gene GmZFP7 of soybean C2H2 type zinc finger protein transcription factor GmZFP7 in the plant.

[0045] Preferably, the plant is selected from soybeans and tobacco.

[0046] In some specific embodiments, regulating isoflavones refers to increasing or decreasing the isoflavone content in the roots, leaves, and seeds of plants.

[0047] Group 2 Examples: New Applications of the Gene GmZFP7 of the Present Invention

[0048] This set of examples provides the application of the gene GmZFP7 with gene accession number Gene ID:100792169 in the regulation of isoflavones.

[0049] In some embodiments, the gene locus number of gene GmZFP7 is Glyma.20G012700; the nucleotide sequence of gene GmZFP7 is shown in SEQ ID NO.1.

[0050] In other embodiments, the gene GmZFP7 regulates isoflavones by activating the expression of the gene GmIFS2, which isoflavone synthase 2, and / or by inhibiting the expression of the gene GmF3H1, which isoflavone-3-hydroxylase 1.

[0051] In a specific embodiment, regulating isoflavones refers to increasing or decreasing the isoflavone content in the plant; more specifically, regulating isoflavones refers to increasing or decreasing the isoflavone content in the roots, leaves, and seeds of the plant.

[0052] Preferably, the plant is selected from soybeans and tobacco.

[0053] Group 3 Examples, Method for Regulating Isoflavones of the Present Invention

[0054] This set of embodiments provides a method for regulating isoflavones. All embodiments in this set share the following common feature: isoflavones are regulated by controlling the level of soybean C2H2 type zinc finger protein transcription factor GmZFP7, whose amino acid sequence is shown in SEQ ID NO.2, and / or by controlling the expression of gene GmZFP7 with gene accession number Gene ID:100792169.

[0055] In a further embodiment, the level of soybean C2H2 type zinc finger protein transcription factor GmZFP7, whose amino acid sequence is shown in SEQ ID NO.2, is regulated by overexpressing, silencing, knocking down, or knocking out the gene GmZFP7, and / or the expression of gene GmZFP7 with gene accession number Gene ID:100792169 is regulated.

[0056] In a specific embodiment, the overexpression refers to: plants transformed with the recombinant overexpression vector PTF101-GmZFP7-GFP or pGGP-GmZFP7 obtained by ligating the gene GmZFP7 to the expression vector PTF101-GFP or pGGP;

[0057] Preferably, the primer sequences for ligating the gene GmZFP7 into the expression vector are shown in SEQ ID NO.5-6;

[0058] The silencing expression refers to the recombinant silencing expression vector pGGP-GmZFP7-RNAi transformed into plants by ligating the RNAi sequence of the gene GmZFP7 into the vector pGGP;

[0059] Preferably, the primer sequences for ligating the RNAi sequence of the gene GmZFP7 into the vector pGGP are shown in SEQ ID NO.7-10;

[0060] The knockout refers to: transforming plants with the JRH0645-GmZFP7 gene editing vector;

[0061] Preferably, the JRH0645-GmZFP7 gene editing vector is constructed by using JRH0645 (CaMV35s:Cas9) as the original vector and inserting the U6 promoter, the gRNA targeting GmZFP7, and the gRNA scaffold in tandem with PCR into the XbaI restriction site; preferably, the target sequence of the gRNA is shown in SEQ ID NO.31.

[0062] Experimental example: Verification of the regulation of isoflavone content by the GmZFP7 protein and its gene in this invention.

[0063] I. Materials and Methods

[0064] 1. Experimental Materials

[0065] Soybean varieties: Luhei No. 2 (LHD), Williams 82

[0066] Ben's Tobacco

[0067] K599 Agrobacterium competent cells (purchased from Zhuangmeng Biotechnology Co., Ltd.)

[0068] Escherichia coli DH5α (purchased from Zhuangmeng Biotechnology Co., Ltd.)

[0069] Trans-T1 Escherichia coli competent cells (purchased from TransGen Biotech Co., Ltd.)

[0070] EHA105 Agrobacterium competent cells (purchased from Zhuangmeng Biotechnology Co., Ltd.)

[0071] ENA105 (psoup) competent cells (purchased from Zhuangmeng Biotechnology Co., Ltd.)

[0072] KOD FX High Fidelity Enzyme

[0073] BAR rapid test strips

[0074] 18% glufosinate (Bayer, Germany)

[0075] The B5 medium basal salts and corresponding vitamins, as well as the MS medium basal salts and corresponding vitamins, were purchased from Beijing Ximeijie Technology Co., Ltd. Disposable sterile plastic dishes were purchased from Beijing Boyuan Hongda Biotechnology Co., Ltd. The plant genomic DNA rapid extraction kit was purchased from Tiangen Biotech Co., Ltd.

[0076] Plant RNA extraction kit purchased from Kingbate Biotechnology Co., Ltd.

[0077] The reverse transcription kit was purchased from TransGen Biotech Ltd.

[0078] AS (acetylsalicylic acid), MES, MgCl2

[0079] Rapid Protein Extraction Kit (Kangwei Biotechnology Co., Ltd.)

[0080] Dual-Glo Luciferase Assay System Kit

[0081] 2. Main instruments and equipment

[0082] PCR amplification instrument (Bio-RAD), electrophoresis apparatus (DYY-6C), benchtop constant temperature shaker (THZ-D), high-speed refrigerated centrifuge (SiGMR), high-speed benchtop centrifuge (SIGMR 3-30K), gel imaging analyzer (Tanon 3500), constant temperature incubator (LRH-250A), handheld fluorescence spectrometer and Bio-Rad MyiQ monochrome fluorescence real-time quantitative PCR instrument, LUYOR-3260RB flashlight fluorescent protein observation microscope and Bio-Rad MyiQ monochrome fluorescence real-time quantitative PCR instrument, SYNERGY H1 full-function microplate reader.

[0083] 3. Experimental Methods

[0084] 1) Gene cloning

[0085] RNA was extracted from the first true leaf of *L. hirsuta*, and the leaf RNA was reverse transcribed into cDNA using a reverse transcription kit. Using the cDNA as a template and GmZF-F / R primers, the complete coding region (CDs) of the GmZF1 transcription factor was cloned for subsequent vector construction. Simultaneously, genomic DNA was extracted from *L. hirsuta* using a plant genomic DNA extraction kit according to the instructions. Using this DNA as a template, the promoter regions 1500-2000 bp upstream of the genes IFS2 (Glyma.13G173500) and F3H1 (Glyma.02G048400) were cloned.

[0086] 2) Carrier construction

[0087] Soybean hairy root experiment used the pGFPGUSplus (pGGP) plant expression vector as the original vector. The GUS gene fragment on the vector was replaced with the desired target gene fragment by double digestion with BglII and BstEII. Overexpression vectors pGGP-GmZFP7 (pGGP-GmZFP7) for hairy root transformation were constructed. Figure 1) and the repressive expression vector pGGP-GmZFP7-RNAi ( Figure 2 ).

[0088] The dual-luciferase assay in tobacco used PTF101-GFP and pGreen-0800II as the original vectors to construct the GmZFP7 plant overexpression vector PTF101-GmZFP7-GFP. Figure 3 ) and signal carrier pGmIFS2 / pGmF3H1-LUC signal carrier ( Figure 4 ).

[0089] The soybean gene editing vector JRH0645 (CaMV35s:Cas9) was used as the original vector. The U6 promoter, gRNA targeting GmZFP7, and gRNA scaffold were tandemly inserted into the XbaI restriction site using PCR to construct the JRH0645-GmZFP7 gene editing vector. Figure 5 The U6 promoter and gRNA scaffold are existing element sequences in the original vector JRH0645 (CaMV35s:Cas9).

[0090] The relevant primers are shown in Table 1.

[0091] Table 1

[0092]

[0093] 3) Transformation of Agrobacterium K599 competent cells

[0094] Add the constructed plasmid to 100 μL of freshly frozen-thawed K599 competent cells, mix gently, and transfer to an electroporation cuvette for electroporation transformation. Add 500 μL of antibiotic-free YEP liquid medium, pipette to mix 2-3 times, transfer the mixture to a 1.5 mL centrifuge tube, and incubate at 200 rpm and 28°C for 3 hours. Spread an appropriate amount of bacterial culture onto YEP solid plates containing the appropriate antibiotic and incubate at 28°C for 36-48 hours.

[0095] 4) Hairy root induction culture

[0096] (a) Seed sterilization: Select healthy seeds in petri dishes and sterilize them using chlorine gas (add 80 mL of sodium hypochlorite and 5 mL of concentrated hydrochloric acid to the beaker). Place the petri dishes and soybean seeds together in a desiccator and sterilize them in a sealed container for about 16-18 hours.

[0097] (b) Seed germination: The sterilized seeds were planted in germination medium and germinated at 25°C under 16h light / 8h darkness.

[0098] (c) Preparation of bacterial culture: The preserved bacterial culture was activated twice in YEP liquid medium at 28°C and 200 rpm until the OD600 was about 0.6-0.8.

[0099] (d) Obtaining explants: Take soybean seeds that have germinated for 4-7 days, cut them off 1-2 mm from the hypocotyl, cut the cotyledons in half, and remove the apical bud. Make 5-7 incisions at the cotyledon node with a blade to obtain the explants.

[0100] (e) Explant infection: Centrifuge the bacterial culture (6000 rpm, 10 min) and readjust the OD600 to 0.6-0.8 with liquid co-culture medium, and infect the cotyledons for 15-20 min.

[0101] (f) Co-culture: Transfer the cotyledons to a solid co-culture medium lined with sterile filter paper. Incubate at 25°C in the dark for 3 days. (g) Root induction culture: After co-culture, wash the explants 3-5 times with double-distilled water containing antibiotics, then transfer them to a root induction medium and culture at 25°C under 16h light / 8h dark conditions.

[0102] (h) Hairy root detection: The induction culture dish was irradiated with a fluorescence microscope. The roots that showed green fluorescence in the field of view were positive roots. The positive and negative roots were counted and sampled.

[0103] 5) Transformation of Agrobacterium EHA105 and EHA105(pSoup) competent cells and transient transformation in tobacco

[0104] Add plasmid DNA to 100 μL of competent cells, mix well, and perform heat shock transformation sequentially: 5 minutes on ice, 5 minutes in liquid nitrogen, 5 minutes in a 37°C water bath, and 5 minutes on ice. Add antibiotic-free LB broth and incubate at 28°C with shaking for 2-3 hours. Centrifuge at 5000 rpm for 1 minute to collect the bacteria, collect 100 μL of supernatant, gently pipette the resuspended bacterial block, spread it onto an LB plate responsive to antibiotics, and incubate upside down at 28°C for 2-3 days.

[0105] Newly activated Agrobacterium monoclonal antibodies were inoculated into YEP containing the appropriate antibiotic and incubated overnight at 28°C and 200 rpm. When the bacterial culture showed an OD of [missing value]... 600 When the OD value is between 0.6 and 1.0, collect Agrobacterium by centrifugation at 1000g for 5 min. Gently resuspend in 2 mL of induction medium, then centrifuge again to collect the bacterial culture. Resuspend the resulting precipitate in 1 mL of induction medium. Measure the OD value after incubation at room temperature for 1–4 hours. Prepare the infection solution according to experimental needs. Inject the infection solution into leaves of *Nicotiana benthamiana* that have grown for 6–8 weeks using a syringe, treat in the dark for 12 hours, and then incubate in an incubator for 48–72 hours.

[0106] 6) Detection of fluorescence signals Luc and Ren in tobacco leaves

[0107] 48-72 hours after tobacco injection, observe the injection site for GFP fluorescence signal using a fluorescent protein microscope. Take 0.5g of tobacco leaves with strong GFP signal, grind them with liquid nitrogen, and extract cytoplasmic soluble protein from the leaves. Place the extracted protein on ice for later use. Take the Dual-Glo Luciferase Assay System kit stored at -20℃ and thaw it on ice. Mix the Dual-Glo Luciferase Substrate and Dual-Glo Luciferase Buffer from the kit and place it on ice to prepare Buffer-Luc. Mix the Dual-Glo Stop&Glo Buffer and Dual-Glo Stop&Glo Substrate and place them on ice to prepare Buffer-Ren. Add the cytoplasmic protein extracted from tobacco and Buffer-Luc to an ELISA plate, mix well with a pipette, and then detect Luc activity using a SYNERGY H1 full-function ELISA reader. After the Luc detection is complete, add 75uL of Buffer-Ren to the above mixture and mix well to terminate the Luc reaction. Start the Ren reaction and detect the Luc activity.

[0108] 7) Soybean genetic transformation

[0109] Seed sterilization: Select the soybean variety Williams82 and sterilize it using chlorine. After sterilization, place the petri dish containing the seeds in a laminar flow hood and blow for 15 minutes to remove residual chlorine.

[0110] Seed germination: Sterilized soybean seeds were planted with the hilum facing down in the germination medium. After sealing, the seeds were cultured at 25°C for 16 hours under light and 8 hours in darkness.

[0111] Preparation of bacterial suspension: Take 40-60 μL of Agrobacterium suspension and spread it evenly on a solid culture medium containing YEP+Rif+carrier and the corresponding antibiotic. Incubate upside down in a dark incubator at 28℃ for 48 h. After activation, resuspend in double-distilled water and spread evenly on a culture medium containing the corresponding antibiotic. Seal and incubate upside down in a dark incubator at 28℃ for 24 h. Resuspend in resuspending solution until OD. 600 Values ​​of 0.6-0.8 are used for subsequent infection.

[0112] Preparation of cotyledonary node explants: Remove the seed coat, separate the two cotyledons, gently remove the cotyledons with a knife, and make a few light cuts at the junction of the cotyledonary nodes. Place the treated seeds into the bacterial solution and incubate for 1.5 hours.

[0113] Co-culture: Soak the prepared cotyledonary explants in the prepared bacterial solution for 1.5 hours. Place the soaked cotyledonary explants with the convex side down on CCM medium lined with sterile filter paper, seal and incubate in the dark at 22°C for 5 days.

[0114] Recovery induction culture: After 5 days of co-culture, remove the explants and wash them 4-5 times with liquid induction medium free of sucrose and agar. Blot the surface dry with filter paper and insert them into the induction medium at a 45° angle. Seal and store in a 25°C incubator with 16 hours of light followed by 8 hours of darkness for 7 days.

[0115] Selection and induction culture: Take soybean explants after recovery culture, remove excessively long hypocotyls and clustered shoots, cut off part of the radicle, leaving about 0.5 cm, and insert them into the selection medium at a 45° angle. Seal and place in a 25℃ culture room, cultured for 21 days under 16h light and 8h darkness conditions.

[0116] Elongation subculture: Explants cultured for 21 days were selected, and part of the hypocotyl was removed. The explants were then inserted at a 45° angle into the elongation medium. The explants were cultured in a sealed container at 25°C under 16 hours of light and 8 hours of darkness. The initial elongation culture lasted 21 days, after which subculture could be repeated every 15 days, for a total of 2-3 times. The transgenic seedlings after elongation subculture were then washed and transplanted.

[0117] 8) Bar identification of transgenic plants

[0118] Screening was mainly conducted using two methods: BAR test strips and glufosinate spraying or smearing.

[0119] BAR rapid test strip: Take a small amount of leaf fragments into a 1.5mL centrifuge tube, add a small amount of water to break up the leaf tissue, insert the BAR test strip into the leaf fragments and wait for about 3 minutes. When a strip appears at the end of the test strip far from the liquid surface, the liquid surface is full of the test strip. At this time, you can observe the test results.

[0120] Glufosinate spraying or application: Dilute 18% glufosinate 1000 times and apply it to the leaf surface and mark it. Observe after 3-5 days.

[0121] 9) PCR sequencing identification of gene-edited plants

[0122] For gene-edited plants, in addition to detection of the Bar gene, the main detection methods used were PCR amplification and sequencing of the CRISPR-Cas9 gene. Leaves from the flowering stage of transgenic plants were collected, and whole-genome DNA was extracted from the leaves using a plant whole-genome DNA extraction kit manufactured by Tiangen Biotech. Cas9 detection and downstream gene editing detection were performed on the CRISPR gene-edited plants. PCR primers are shown in Table 2.

[0123] Table 2. Primers for detecting CRISPR-edited plants

[0124]

[0125]

[0126] 10) Extraction and detection methods of isoflavones

[0127] The tissue to be tested was ground into powder using a cyclone mill (mortar). 0.02 g of the powder was weighed into a 2 mL centrifuge tube, and 1 mL of an extract containing 70% (v / v) ethanol and 0.1% (v / v) acetic acid was added. The mixture was then shaken and mixed for 12 h. The completely mixed extract was centrifuged at 2700 g for 10 minutes at 4 °C, and the supernatant was collected. The supernatant was filtered through a 0.2 μm filter (YMC, Kyoto, Japan). The 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 I.D., S-5 μm). Mobile phases A and B consist of 0.1% acetic acid and acetonitrile dissolved in distilled water, respectively. The solvent flow rate is 1.0 mL / min. -1 The injection volume was 10 μL, using a 70-minute linear gradient of 13-30% acetonitrile (v / v). The UV detector wavelength was set to 260 nm, and the column temperature was set to 35 °C. The soybean isoflavone standard sample consisted of 12 components: genistein, daidzein, daidzein, malonyl genistein, malonyl daidzein, acetyl daidzein, acetyl genistein, acetyl daidzein, daidzein, daidzein, and daidzein, with a concentration of 200 μg / mL. -1 Equal volumes of each standard sample were mixed to prepare a mixed standard sample, which was then stored at -20℃ for later use. Qualitative analysis was performed based on the retention time and maximum absorption spectrum of the 12 isoflavone standard samples. The ultraviolet absorption value at a wavelength of 260 nm was used as the standard, and the content of each isoflavone component, aglycone and total content in the sample was calculated according to the method of Sun Junming et al. (2011) (Sun J, Sun B, Han F, Yan S, Yang H and Kikuchi A. Rapid HPLC method for determination of 12 isoflavone components in soybean seeds. AgriSci China, 2011, 10(1): 101-105.).

[0128] 11) qRT-PCR analysis

[0129] Real-time PCR was used to detect the expression levels of silenced and overexpression vector genes in soybean hairy roots, using the GmActin-11-like gene as an internal reference. The procedure was performed according to the TaKaRa SYBR Premix Ex taq II instruction manual. The primer sequences used are shown in Table 3. Gene expression levels were calculated using a 2-1... -ΔΔCT Calculation by method.

[0130] Table 3

[0131]

[0132] II. Experimental Results

[0133] 1. Overexpression and silencing of GmZFP7 significantly altered the isoflavone content in soybean hairy roots.

[0134] In Luhei soybean '2', overexpression of GmZFP7 via the pGGP-GmZFP7 vector increased the relative expression level of GmZFP7 in transgenic hair roots by 1-2420 times, and the total isoflavone content increased by 13%-29%. Silencing GmZFP7 via the pGGP-GmZFP7-RNAi vector reduced the relative expression level of GmZFP7 by 81-88%, and the total isoflavone content decreased by 18-28%. This indicates that overexpression and silencing of GmZFP7 significantly altered the total isoflavone content in soybean hair roots. Figure 6 ).

[0135] 2. GmZFP7 significantly activates the expression of isoflavone synthase 2 (GmIFS2), a node enzyme in the isoflavone synthesis pathway, while inhibiting the expression of flavanone-3-hydroxylase 1 (GmF3H1), which competes for a common substrate.

[0136] Using CaMV35s:Ren as an internal control and PTF101-GFP vector as a control, experiments were conducted to determine the regulation of GmIFS1, GmIFS2, and GmF3H1 promoters by detecting Luc enzyme activity in the pGreen vector. Results showed that GmZFP7 expression did not significantly change the promoter activity of GmIFS1, increased the promoter activity of GmIFS2 by 3.3-fold, while decreased the promoter activity of GmF3H1 by 60%. Figure 7 The results showed that GmZFP7 increases isoflavone content by activating the expression of isoflavone synthase 2 (GmIFS2), a key node enzyme in the isoflavone synthesis pathway, while inhibiting the expression of flavanone-3-hydroxylase 1 (GmF3H1), which competes for a common substrate.

[0137] 3. The total isoflavone phenotype of stable soybean plants showed that modification of GmZFP7 could significantly alter the isoflavone content in soybean leaves and seeds.

[0138] 3.1 Obtaining and identifying plants overexpressing GmZFP7

[0139] The GmZFP7 overexpression vector PTF101-GmZFP7-GFP and the control vector PTF101-GFP were transformed into the soybean variety Williams82 using the soybean cotyledon node genetic transformation method. Figure 8 The obtained overexpression lines were screened using Bar test strips, glufosinate smear screening, and GmZFP7 qPCR detection. Three T4 generation overexpression homozygous lines with Bar gene and glufosinate resistance were screened, along with three lines carrying the empty vector PTF101-GFP as controls. The results are as follows: Figure 8 .

[0140] 3.2 Overexpression of GmZFP7 can increase the isoflavone content in soybean leaves and seeds.

[0141] Leaves and seeds of stably inherited transgenic T4 generation plants were selected to detect the gene expression level of GmZFP7 and the total isoflavone content. In the three overexpression lines of GmZFP7 (OE1, OE2, and OE3), the relative expression level of GmZFP7 was 17-125 times higher than the control. Figure 9 A) The expression levels of the GmZFP7 gene varied significantly among the three overexpression lines, possibly due to the different copy numbers of the GmZFP7 gene inserted into the genome. However, all three overexpression lines showed significantly higher expression levels compared to the control line.

[0142] Meanwhile, the total isoflavone content in both leaves and seeds was significantly increased, with an increase of 35%-39% in leaves and a significant increase of 7%-19% in seeds. Figure 9 Based on transient expression experiments in hairy roots and tobacco, we examined the changes in the relative expression levels of GmIFS2 and GmF3H1 in transgenic plants. In the leaves of the three overexpression lines, the relative expression level of GmIFS2 increased by 2.0-2.8 times, while the expression level of GmF3H1 decreased by 40%-80%. Figure 9 D). The above results indicate that GmZFP7 can increase isoflavone content by regulating the metabolic flux of the phenylpropane metabolic pathway by increasing the expression of GmIFS2 in the isoflavone pathway and inhibiting the expression of GmF3H1 in the flavonol pathway.

[0143] 3.3 CRISPR / Cas9-mediated selection of GmZFP7 knockout plants

[0144] Based on the sequence characteristics of GmZFP7, a suitable sgRNA target sequence, GGTTTGAACTTAGACCTTGGTCT (SEQ ID NO.31) (PAM is in red), was designed and screened online using CRISPR-P2.0. Figure 10 A). For T0-T1 generation transgenic edited plants, Bar gene screening and Cas9 gene molecular identification were first performed, followed by individual sowing and harvesting. Figure 10 B), 31 bimodal heterozygous editing materials were screened from three T2 generation gene editing lines, including 4 homozygous mutants with the same editing type, all of which were single-base insertions. Figure 10 C,D).

[0145] 3.4 GmZFP7 knockout significantly reduced isoflavone content in leaves and seeds of transgenic plants.

[0146] Homozygous knockout mutant Gmzfp7 plants, selected from positive T2 generation transgenic plants, were harvested individually. T3 generation plants were selected, and the total isoflavone content and relative expression levels of related genes in leaves and seeds were measured. The total isoflavone content in both leaves and seeds of the Gmzfp7 mutant was significantly reduced. Figure 11 (A, B). qPCR results showed that the expression level of GmZFP7 in the mutant did not change significantly, the expression level of GmIFS2 was significantly decreased, and the expression level of GmF3H1 was significantly increased, consistent with the previous results. This indicates that GmZFP7 can change the metabolic flux of phenylpropane by increasing the expression of GmIFS2 in the isoflavone pathway and inhibiting the expression of GmF3H1 in the flavonol pathway, thereby increasing the total isoflavone content.

Claims

1. Application of soybean C2H2 type zinc finger protein transcription factor GmZFP7, whose amino acid sequence is shown in SEQ ID NO. 2, in regulating soybean isoflavone content.

2. The soybean C2H2 type zinc finger protein transdermal protein according to claim 1, with the amino acid sequence shown in SEQ ID NO.

2. The application of transcription factor GmZFP7 in regulating soybean isoflavone content is characterized by, The nucleotide sequence of the gene GmZFP7, which is the soybean C2H2 type zinc finger protein transcription factor GmZFP7, is shown in SEQ ID NO.

1.

3. Soybean C2H2 type zinc finger protein with an amino acid sequence as shown in SEQ ID NO. 2 according to claim 1 or 2. The application of transcription factor GmZFP7 in regulating soybean isoflavone content is characterized by, The regulation of soybean isoflavone content refers to increasing or decreasing the level of soybean C2H2 type zinc finger protein transcription factor GmZFP7 in the plant, or increasing or decreasing the isoflavone content in soybean plants by overexpressing, silencing, knocking down, or knocking out the gene GmZFP7 of soybean C2H2 type zinc finger protein transcription factor GmZFP7 in the plant.

4. Application of gene GmZFP7, whose nucleotide sequence is shown in SEQ ID NO. 1, in regulating soybean isoflavone content.

5. The application of the gene GmZFP7, with the nucleotide sequence shown in SEQ ID NO. 1 according to claim 4, in regulating soybean isoflavone content, characterized in that... The locus number of the gene GmZFP7 is Glyma .20G012700.

6. The application of the gene GmZFP7, with the nucleotide sequence as shown in SEQ ID NO. 1 according to claim 4 or 5, in regulating soybean isoflavone content, characterized in that... The gene GmZFP7 regulates soybean isoflavone content by activating the expression of the isoflavone synthase 2 gene GmIFS2 or inhibiting the expression of the flavanone-3-hydroxylase 1 gene GmF3H1.

7. A method for regulating the content of soybean isoflavones, characterized in that, Soy isoflavone content can be regulated by controlling the level of soybean C2H2 type zinc finger protein transcription factor GmZFP7 (amino acid sequence as shown in SEQ ID NO.2) or by regulating the expression of gene GmZFP7 (nucleotide sequence as shown in SEQ ID NO.1).

8. The method for regulating the content of soybean isoflavones according to claim 7, characterized in that, The level of soybean C2H2 zinc finger protein transcription factor GmZFP7 (as shown in SEQ ID NO. 2) or the expression of gene GmZFP7 (as shown in SEQ ID NO. 1) can be regulated by overexpressing, silencing, or knocking out the gene GmZFP7.

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