Biosynthetic method for catalytic synthesis of daidzein precursor, daidzein based on daidzein

Abstract

This invention relates to the field of biotechnology, specifically to a biosynthetic method for the synthesis of daidzein precursors and daidzein based on daidzein catalysis. This invention synthesizes daidzein precursors glyceollidinⅠ (GD1) or glyceollidinⅡ (GD2) by co-expressing the genes AtPAL2, GmCHS8, GmCHR5, GmCHI1B1, GmIFS1, GmHID and AtMYB60, which are involved in daidzein synthesis, along with downstream daidzein genes GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH and GmG4DT, or GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH and GmG2DT, in *Nicotiana benthamiana* mediated by *Agrobacterium*. Furthermore, it co-expresses any one or a combination of several of the daidzein synthase genes GmGS1a, GmGS1b, GmGS2a, GmGS2b, GmGS3 / 7, and GmGS3e. This invention utilizes the Benedictine platform, based on daidzein, a key precursor in the biosynthesis of highly accumulated isoflavones, to reconstruct the biosynthetic pathways of the direct precursors of daidzein, GD1 and GD2. It elucidates the daidzein biosynthetic pathway, paving the way for heterologous daidzein biosynthesis and laying a scientific theoretical and technological foundation for the large-scale industrial production and medicinal function research of antibacterial daidzein.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a biosynthetic method for the synthesis of daidzein precursors and daidzein based on daidzein catalysis. Background Technology

[0002] Daidextrin is a class of low-molecular-weight isoflavone phytotoxicants synthesized and accumulated in soybean tissues after microbial infection. Compared with other isoflavones, it exhibits stronger antifungal, antioxidant, and anticancer activities, showing broad application prospects in green pest and disease control and food development. However, daidextrin synthesis in plants requires induction and is extremely low in yield, with a complex chemical structure, which severely hinders the acquisition of sufficient daidextrin through traditional plant extraction and chemical synthesis. Daidextrin is catalyzed by daidzein, a key precursor produced in the upstream pathway of isoflavones. Its complete biosynthetic pathway involves many enzymatic reactions, and the precursor accumulation in plants is low. Some pathway genes have not been characterized, and the catalytic enzyme in the final step of its biosynthesis, daidextrin synthase (GS), has not yet been discovered and verified. These problems severely limit the large-scale production of antibacterial daidextrin using standardized industrial processes and its pharmaceutical development. Therefore, elucidating the complete daidextrin biosynthetic pathway and developing daidextrin biosynthetic methods are prominent challenges that urgently need to be addressed. For microbial synthesis platforms, the de novo synthesis of 15-step enzyme-catalyzed daidzein and the expression of multiple cytochrome P450 enzymes remains a huge challenge. Therefore, finding a suitable heterologous host to elucidate the complete daidzein biosynthesis pathway is crucial. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a biosynthetic method for the synthesis of daidzein precursors and daidzein based on daidzein catalysis, which addresses the shortcomings of the prior art.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0005] Based on the biosynthetic method of daidzein-catalyzed synthesis of daidzein precursors, using Nicotiana benthamiana as the starting plant, daidzein precursors were obtained by co-expressing genes for daidzein synthesis and downstream genes for daidzein synthesis in Nicotiana benthamiana mediated by Agrobacterium.

[0006] The downstream genes for daidzein synthesis are GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, and GmG4DT, or GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, and GmG2DT.

[0007] Specifically, the GenBank accession numbers of GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, GmG4DT, and GmG2DT are as follows: XM_003533533.4, NM_001289198.2, NM_001249591.2, NM_001250005.4, NM_001254257.2, AB434690.1, and NM_001348662.1.

[0008] The gene for synthesizing daidzein consists of 7 genes;

[0009] Specifically, the seven genes are phenylalanine ammonia-lyase gene AtPAL2, chalcone synthase gene GmCHS8, chalcone reductase gene GmCHR5, chalcone isomerase gene GmCHI1B1, isoflavone synthase gene GmIFS1, 2-hydroxyisoflavone dehydratase gene GmHID, and transcription factor AtMYB60.

[0010] Specifically, the GenBank accession numbers for the phenylalanine ammonia-lyase gene AtPAL2, chalcone synthase gene GmCHS8, chalcone reductase gene GmCHR5, chalcone isomerase gene GmCHI1B1, isoflavone synthase gene GmIFS1, 2-hydroxyisoflavone dehydratase gene GmHID, and transcription factor AtMYB60 are, respectively: NM_115186.4, AY237728.1, LC309095.1, NM_001249826.2, AF195798.1, AB154415.1, and NM_100755.3.

[0011] The gene for synthesizing daidzein is expressed via plasmid MGV.

[0012] Specifically, the plasmid MGV is a multi-gene expression vector pEAQ-35S promoter-GmCHIB1-P2A-AtPAL2-T2A-GmHID-NOS terminator-35Spromoter-GmCHS8-P2A-GmCHR5-NOS teminator-35S promoter-GmIFS1-P2A-AtMYB60-NOSteminator.

[0013] Specifically, the genes in the gene expression cassette are linked by 2A peptides, wherein the 2A peptides are any one or a combination of two of the P2A peptides or T2A peptides.

[0014] Specifically, the amino acid sequence of the P2A peptide is shown in SEQ ID NO.1, and the corresponding nucleotide sequence is shown in SEQ ID NO.2; the amino acid sequence of the T2A peptide is shown in SEQ ID NO.3, and the corresponding nucleotide sequence is shown in SEQ ID NO.4.

[0015] The downstream genes of daidzein synthesis are expressed through the expression vector pEAQ-HT containing the downstream genes of daidzein synthesis, namely through pEAQ-GmI2'H, pEAQ-GmIFR, pEAQ-GmVR, pEAQ-GmPTS1, pEAQ-GmD6aH, pEAQ-GmG4DT and pEAQ-GmG2DT.

[0016] The daidzein precursor is glyceollidin I (GD1) or glyceollidin II (GD2).

[0017] Based on the biosynthetic method of daidzein catalyzed by daidzein, daidzein was synthesized by using Nicotiana benthamiana as the starting plant and expressing genes for daidzein synthesis, downstream genes for daidzein synthesis, and daidzein synthase genes in Nicotiana benthamiana in an Agrobacterium-mediated manner.

[0018] The gene for synthesizing daidzein is expressed via plasmid MGV.

[0019] Specifically, the plasmid MGV is a multi-gene expression vector containing three gene expression cassettes that express seven genes;

[0020] Specifically, the seven genes are phenylalanine ammonia-lyase gene AtPAL2, chalcone synthase gene GmCHS8, chalcone reductase gene GmCHR5, chalcone isomerase gene GmCHI1B1, isoflavone synthase gene GmIFS1, 2-hydroxyisoflavone dehydratase gene GmHID, and transcription factor AtMYB60.

[0021] The daidzein includes daidzein 1, daidzein 2, daidzein 3, 13-epi-daidzein 3, and daidzein 7.

[0022] Among them, daidzein 7 is a novel daidzein with the following chemical structural formula:

[0023]

[0024] The downstream genes for daidzein synthesis are GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, GmG4DT, or GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, GmG2DT.

[0025] The soybean synthase gene includes any one or a combination of several of GmGS1a, GmGS1b, GmGS2a, GmGS2b, GmGS3e / 7, and GmGS3.

[0026] Specifically,

[0027] (1) When the downstream gene for daidzein synthesis is GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, or GmG4DT, and the daidzein synthase gene is GmGS1a or GmGS1b, the daidzein is daidzein 1.

[0028] (2) When the downstream gene for daidzein synthesis is GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, or GmG2DT, and the daidzein synthase gene is GmGS2a or GmGS2b, the daidzein is daidzein 2.

[0029] (3) When the downstream genes for daidzein synthesis are GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, and GmG2DT, and the daidzein synthase gene is GmGS3e / 7, the daidzein is 13-epi-daidzein3 and daidzein7.

[0030] (4) When the downstream genes for daidzein synthesis are GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, and GmG2DT, and the daidzein synthase gene is GmGS3, the daidzein is daidzein 3.

[0031] Beneficial effects:

[0032] (1) Based on the Nicotiana benthamiana platform of daidzein, a key precursor for the biosynthesis of high-accumulation isoflavones, this invention has explored and verified the function of the gene encoding daidzein synthase. Combined with chemical characterization methods, the complete pathway of daidzein biosynthesis has been elucidated, laying a scientific theoretical and technical foundation for the large-scale industrial production and medicinal function research of antibacterial daidzein.

[0033] (2) This invention successfully reconstructed the biosynthetic pathways of the direct precursors of daidzein, GD1 and GD2, in Tobacco Benzoinus, and identified the functions of key enzymes in the daidzein biosynthetic pathway from soybean, laying the foundation for the subsequent screening and identification of unknown daidzein synthases.

[0034] (3) This invention screened out 6 daidzein synthases and synthesized four known daidzeins and one novel daidzein, paving the way for heterologous biosynthesis of daidzeins. Attached Figure Description

[0035] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0036] Figure 1 This is a diagram of the predicted daidzein synthesis pathway.

[0037] Figure 2 This study reconstructs the biosynthetic pathway of daidzein precursors. MGV is a multi-gene vector for daidzein synthesis, containing the genes AtPAL2, GmCHS8, GmCHR5, GmCHI1B1, GmIFS1, GmHID, and AtMYB60. DHP stands for 3,9-Dihydroxypterocarpan; GD1 for glyceollidin I; GD2 for glyceollidin II; GD1-3-gly for 3-glucose-glyceollidin I; and GD2-3-gly for 3-glucose-glyceollidin II. Figure 3 These are mass spectra of intermediates and precursors in the biosynthesis of daidzein. In the spectrum, A represents the primary and secondary mass spectra of DHP; B represents the secondary mass spectra of GD1-3-gly and GD2-3-gly; and C represents the secondary mass spectra of GD1 and GD2. MS indicates primary mass spectra, MS2 indicates secondary mass spectra, and RT indicates retention time.

[0038] Figure 4 The NMR spectrum of the purified daidzein precursor GD1-3-gly is shown. A is the proton NMR spectrum; B is the carbon NMR spectrum.

[0039] Figure 5 NMR data for the purified daidzein precursor GD1-3-gly.

[0040] Figure 6 The NMR spectrum of the purified daidzein precursor GD2-3-gly is shown. A is the proton NMR spectrum; B is the carbon NMR spectrum.

[0041] Figure 7 NMR data for the purified daidzein precursor GD2-3-gly.

[0042] Figure 8 The HPLC chromatograms show the hydrolysis of GD1-3-gly and GD2-3-gly by β-glucosidase. GC1 represents: MGV + GmI2'H + GmIFR + GmVR + GmPTS1 + GmD6aH + GmG4DT; GC2 represents: MGV + GmI2'H + GmIFR + GmVR + GmPTS1 + GmD6aH + GmG2DT.

[0043] Figure 9The image shows the liquid chromatograms of the screening results for functional soybean synthases. GC1 represents: MGV+GmI2'H+GmIFR+GmVR+GmPTS1+GmD6aH+GmG4DT; GC2 represents: MGV+GmI2'H+GmIFR+GmVR+GmPTS1+GmD6aH+GmG2DT; EIC represents the extracted ion chromatogram.

[0044] Figure 10 This is a phylogenetic tree of daidzein synthase and its homologous sequences.

[0045] Figure 11 To identify the obtained daidzein biosynthesis pathway.

[0046] Figure 12 This is a secondary mass spectrum of the synthesized product obtained through separation and purification.

[0047] Figure 13 , 15 NMR spectra 17, 19, 21, 23, 25, 27, and 29 are the NMR spectra of the synthesized products obtained after separation and purification. Among them, A is the proton NMR spectrum and B is the carbon NMR spectrum.

[0048] Figure 14 , 16 18, 20, 22, 24, 26, 28, and 30 are the NMR data of the synthesized products obtained through separation and purification, respectively. Detailed Implementation

[0049] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0050] In the following examples, the experimental materials and solution reagents are as follows:

[0051] (1) Source of materials:

[0052] Plant materials: The seeds of *Tobacco Benedict* were preserved in our laboratory; the soybean seeds were from the soybean variety William 82 from Nanjing Agricultural University.

[0053] Vector: The tobacco expression empty vector pEAQ-HT was preserved in our laboratory.

[0054] Strains: DH5α competent cells were purchased from Shenzhen Kangti Biomedical Technology Co., Ltd., for gene cloning. LBA4404 competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd., for transient tobacco transformation.

[0055] (2) Solution formulation

[0056] LB medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride, for the culture of Escherichia coli and Agrobacterium.

[0057] Antibiotics: Prepare a 50 mg / mL kanamycin sulfate solution with a working concentration of 50 mg / L; prepare a 50 mg / mL rifampicin solution with a working concentration of 50 mg / L for the culture of Escherichia coli and Agrobacterium.

[0058] 50×TAE electrophoresis buffer: 242 g / L Tris, 18.61 g / L disodium ethylenediaminetetraacetate, 57.1 mL / L glacial acetic acid, working concentration 1×, used for gene cloning and vector construction.

[0059] 50% glycerol solution: Glycerol and water are mixed evenly in a 1:1 ratio. The working concentration is 15% to 30%, which is used for the preservation of bacterial strains.

[0060] Agrobacterium permeate: 10 mM / L MgCl2, 10 mM / L morpholine ethanesulfonic acid and 0.15 mM / L acetylsyl ketone, used for transient conversion of tobacco.

[0061] In the following embodiments, the primer sequence information and gene information used are shown in Table 1 and Table 2, respectively.

[0062] Table 1 Primer sequences used in this invention

[0063]

[0064]

[0065]

[0066]

[0067] Table 2 Gene information used in this invention

[0068]

[0069]

[0070] In the following embodiments, the extraction and detection methods of the tobacco leaf compounds are as follows:

[0071] (1) Sample preparation: Weigh 10 mg dry weight (DW) of tobacco leaves infected with Agrobacterium, dissolve in 400 μL of 80% methanol, sonicate at room temperature for 45 min, centrifuge at 12000 rpm for 10 min, take 300 μL into a new centrifuge tube, centrifuge at 12000 rpm for 5 min, and take 200 μL into a sample vial for sample loading.

[0072] (2) Preparation of mobile phase: Phase A: Take an appropriate amount of formic acid and dilute it with water to a concentration of 0.1%. Take 1L of 0.1% formic acid-water solution and sonicate for 30min. Phase B: Take 1L of acetonitrile and sonicate for 30min.

[0073] (3) Chromatographic analysis conditions: The chromatographic column used was a Phenomenex Kinetex C18 column (100 mm × 2.1 mm, 1.7 μm); injection volume: 2 μL; flow rate: 0.3 mL / min; column temperature: 35℃; mobile phase: phase A: 0.1% formic acid-water solution, phase B: acetonitrile; detection time: 11.6 min. Elution gradient: 0-0.8 min, 0%-2% B; 0.8-1.0 min, 2%-25% B; 3.0-6.0 min, 25%-35% B; 6.0-7.0 min, 35%-100% B; 10.0-10.2 min, 100%-2% B. Ion source used: electrospray ionization (ESI); scanning mode: positive ion scan, negative ion scan.

[0074] Example 1: Reconstruction of the daidzein precursor biosynthesis pathway

[0075] 1. Gene cloning

[0076] Based on the hypothesized synthetic pathways of the direct precursors of daidzein, glyceollidin I (GD1) and glyceollidin II (GD2) (… Figure 1 Seven downstream genes for daidzein synthesis derived from soybean were cloned: isoflavone 2'-hydroxylase GmI2'H, isoflavone reductase GmIFR, vesitone reductase GmVR, stigmine synthase GmPTS1, 3,9-dihydroxystigmine 6a-monooxygenase GmD6aH, glycine 4-dimethylallyltransferase GmG4DT, and glycine 2-dimethylallyltransferase GmG2DT. The plant expression vector pEAQ-HT was constructed for transient expression in tobacco. Primers used and information on the cloned genes are shown in Tables 1 and 2, respectively. The specific experimental procedure is as follows.

[0077] 100 mg of different tissues, such as roots, stems, and leaves, were collected from soybeans. RNA was extracted using a rapid universal plant RNA extraction kit (purchased from Huayueyang Biotechnology Co., Ltd.), and then used a reverse transcription kit. One-Step gDNARemoval and cDNA Synthesis SuperMix (purchased from Beijing TransGen Biotech Co., Ltd.) was used to reverse transcribe cDNA. Using this cDNA as a template, the target genes (GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, GmG4DT, and GmG2DT) were cloned using high-fidelity DNA polymerase 2xPhantaMax Master Mix (purchased from Nanjing Novizan Biotech Co., Ltd.) and corresponding upstream and downstream primers. After agarose gel electrophoresis, the corresponding PCR amplification products were recovered and purified using a QuickGel Extraction Kit (purchased from Beijing TransGen Biotech Co., Ltd.) to obtain the corresponding target gene fragments.

[0078] The reverse transcription reaction system and procedure, and the PCR amplification reaction system and procedure are as follows:

[0079] Reverse transcription reaction system: RNA 50ng-5μg, Oligo(dT) 1μL, 2X TS Reaction Mix 10μL, RT / RI Enzyme Mix 1μL, gDNA Remover 1μL, RNase-free Water to 20μL; Reverse transcription program: 42℃ for 30min, 85℃ for 5min. After completion, store at -20℃.

[0080] PCR amplification reaction system (50 μl): 2×PCR mix 25 μL, Primer-F (10 μM) 1.5 μL, Primer-R (10 μM) 1.5 μL, ddH2O 20 μL, template 2 μL; PCR amplification program: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 55℃ for 15 s (this step can be adjusted according to different situations), 72℃ for 1 min (this step can be adjusted according to different situations), for 35 amplification cycles; finally, 72℃ extension for 5 min.

[0081] 2. Construction of single-gene vectors

[0082] The pEAQ-HT empty vector was double-digested with Xho I and Age I restriction endonucleases (purchased from New England Biolabs). After agarose gel electrophoresis, the linearized vector was recovered from the gel and ligated into single fragments using the ClonExpress Ultra One Step Cloning Kit V2 (purchased from Nanjing Novizan Biotechnology Co., Ltd.) via homologous recombination. The ligation system was as follows: 1 μL of linearized vector (the amount can be adjusted according to the DNA concentration), 1 μL of target gene fragment (the amount can be adjusted according to the DNA concentration), 5 μL of 2×CE Mix, and 3 μL of ddH2O. After mixing, the mixture was incubated at 50℃ for 5 min, then cooled to 4℃ or immediately placed on ice to obtain the corresponding ligation products.

[0083] The ligation products were then mixed with *E. coli* DH5α competent cells, incubated on ice for 30 min, followed by heat shock at 42°C for 1 min, and immediately placed on ice. After 2 min, 500 μL of LB medium was added, and the cells were incubated at 37°C for 1 h at 200 rpm. The cells were collected at the bottom of the tube by centrifugation at 5000 rpm for 1 min, resuspended in 100 μL of LB medium, and evenly spread onto plates containing antibiotics. The cells were incubated overnight at 37°C. Colony PCR identification confirmed the corresponding positive strains. Plasmids were extracted using the HiPure Plasmid MiniPrep Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.) and sequenced, yielding the correctly sequenced plasmids pEAQ-GmI2'H, pEAQ-GmIFR, pEAQ-GmVR, pEAQ-GmPTS1, pEAQ-GmD6aH, pEAQ-GmG4DT, and pEAQ-GmG2DT.

[0084] 3. Construction of the multi-gene vector MGV for daidzein synthesis

[0085] (1) First, single-gene expression vectors pEAQ-AtPAL2, pEAQ-GmCHS8, pEAQ-GmCHR5, pEAQ-GmCHI1B1, pEAQ-GmIFS1, pEAQ-GmHID, and pEAQ-AtMYB60 were constructed, as described in the section on single-gene vector construction. Primer information is shown in Table 1 (numbers 15-28), and gene information is shown in Table 2. Specifically, AtPAL2 is the phenylalanine ammonia-lyase gene, GmCHS8 is the chalcone synthase gene, GmCHR5 is the chalcone reductase gene, GmCHI1B1 is the chalcone isomerase gene, GmIFS1 isoflavone synthase gene, GmHID is the 2-hydroxyisoflavone dehydratase gene, and AtMYB60 is a transcription factor.

[0086] (2) Based on the above single-gene expression vectors, construct multi-gene expression vectors (single expression cassettes), including pEAQ-GmCHS8-P2A-GmCHR5 (MGV1), pEAQ-GmIFS1-P2A-AtMYB60 (MGV2), and pEAQ-GmCHIB1-P2A-AtPAL2-T2A-GmHID (MGV3).

[0087] MGV1 construction method: The pEAQ-HT empty vector was double-digested with NEB's Xho I and Age I restriction endonucleases. After agarose gel electrophoresis, the linearized vector after digestion was recovered by gel electrophoresis. The two gene fragments were amplified using the constructed single-gene expression vectors pEAQ-GmCHS8 and pEAQ-GmCHR5 as templates.

[0088] Specifically, using pEAQ-GmCHS8 as a template, primers 17 and 29 and 2×Phanta MaxMaster Mix of Novizan were used to amplify the first target gene sequence GmCHS8-P2A-infusion. The amplification reaction system was the same as that used for the construction of the single gene vector mentioned above.

[0089] Using pEAQ-GmCHR5 as a template, primers 20 and 30-31 and 2×Phanta MaxMaster Mix of Novizan were used to amplify the second target gene sequence in two steps using overlap-PCR. The reaction system is as follows:

[0090] Step 1: Mix 0.2 μL of P2A-GmCHR5-F primer, 0.2 μL of pEAQ-GmCHR5-R primer, 5 μL of 2×PhantaMax Master Mix, 0.5 μL of plasmid DNA, and 4.1 μL of ddH2O. Follow the PCR amplification program as follows: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 55℃ for 15 s (this step can be adjusted according to different situations), 72℃ for 1 min (this step can be adjusted according to different situations), for 10 amplification cycles; finally, extend at 72℃ for 5 min, and then use it as a template for the next reaction.

[0091] Step 2: Mix 10 μL of the reaction solution from the previous step, 1 μL of P2A-F primer, 1 μL of pEAQ-GmCHR5-R primer, 25 μL of 2×Phanta Max Master Mix, and 13 μL of ddH2O. Then, follow the PCR amplification program: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 55℃ for 15 s (the temperature can be adjusted according to different situations), 72℃ for 1 min (the time can be adjusted according to different situations), for 30 amplification cycles; finally, extend at 72℃ for 5 min.

[0092] After recovering the above DNA fragments, multiple fragments were ligated using the homologous recombination method with Novizan's ClonExpress Ultra One Step Cloning Kit V2. The ligation system was as follows: 1 μL of linearized vector (the amount can be adjusted according to the DNA concentration), 1 μL each of the target gene fragments (the amount can be adjusted according to the DNA concentration), 5 μL of 2×CE Mix, and ddH2O to a final volume of 10 μL. After mixing, the mixture was incubated at 50°C for 15 min, then cooled to 4°C or immediately placed on ice to obtain the corresponding ligation products.

[0093] The ligation product was then mixed with *E. coli* DH5α competent cells, incubated on ice for 30 min, followed by heat shock at 42°C for 1 min, and immediately placed on ice. After 2 min, 500 μL of LB medium was added, and the cells were incubated at 37°C for 1 h at 200 rpm. The cells were collected at the bottom of the tube by centrifugation at 5000 rpm for 1 min, resuspended in 100 μL of LB medium, and evenly spread onto plates containing antibiotics. The cells were incubated overnight at 37°C. Colony PCR identification confirmed the corresponding positive strain. Plasmids were extracted using the HiPure Plasmid MiniPrep Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.) and sequenced, yielding the correctly sequenced plasmid MGV1: pEAQ-GmCHS8-P2A-GmCHR5.

[0094] MGV2 construction method: Same as MGV1 method, with templates pEAQ-GmIFS1 and pEAQ-AtMYB60. The first target gene amplification primers are numbered 23 and 32, and the second target gene amplification primers are numbered 27, 30 and 33. The final plasmid MGV2 is obtained: pEAQ-GmIFS1-P2A-AtMYB60.

[0095] MGV3 construction method: The pEAQ-HT empty vector was double-digested with NEB's Xho I and Age I restriction endonucleases. After agarose gel electrophoresis, the linearized vector after digestion was recovered by gel electrophoresis. The three gene fragments were amplified using the constructed single-gene expression vectors pEAQ-GmCHIB1, pEAQ-AtPAL2 and pEAQ-GmHID as templates.

[0096] Specifically, using pEAQ-GmCHIB1 as a template, primers 21 and 34 and 2×Phanta MaxMaster Mix of Novizan were used to amplify the first target gene sequence GmCHIB1-P2A-infusion, and the amplification reaction system was the same as above.

[0097] Using pEAQ-AtPAL2 as a template, primers 30 and 35-36 and 2×Phanta MaxMaster Mix of Novizan were used to amplify the second target gene sequence in two steps using overlap-PCR. The reaction system is as follows:

[0098] Step 1: Mix 0.2 μL of P2A-AtPAL2-F primer, 0.2 μL of AtPAL2-T2A-R primer, 5 μL of 2×PhantaMax Master Mix, 0.5 μL of plasmid DNA, and 4.1 μL of ddH2O. Follow the PCR amplification program as follows: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 55℃ for 15 s (this step can be adjusted according to different situations), 72℃ for 1 min (this step can be adjusted according to different situations), for 10 amplification cycles; finally, extend at 72℃ for 5 min, and then use it as a template for the next reaction.

[0099] Step 2: Mix 10 μL of the reaction solution from the previous step, 1 μL of P2A-F primer, 1 μL of AtPAL2-T2A-R primer, 25 μL of 2×Phanta Max Master Mix, and 13 μL of ddH2O. Then, follow the PCR amplification program: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 55℃ for 15 s (the temperature can be adjusted according to different situations), 72℃ for 1 min (the time can be adjusted according to different situations), for 30 amplification cycles; finally, extend at 72℃ for 5 min.

[0100] Using pEAQ-GmHID as a template, primers 26 and 37-38, and 2×Phanta MaxMaster Mix of Novizan, the third target gene sequence was amplified in two steps using overlap-PCR. The reaction system is as follows:

[0101] Step 1: Mix 0.2 μL of T2A-GmHID-F primer, 0.2 μL of pEAQ_GmHID-R primer, 5 μL of 2×PhantaMax Master Mix, 0.5 μL of plasmid DNA, and 4.1 μL of ddH2O. Follow the PCR amplification program as follows: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 55℃ for 15 s (this step can be adjusted according to different situations), 72℃ for 1 min (this step can be adjusted according to different situations), for 10 amplification cycles; finally, extend at 72℃ for 5 min, and then use it as a template for the next reaction.

[0102] Step 2: Mix 10 μL of the reaction solution from the previous step, 1 μL of T2A-F primer, 1 μL of pEAQ_GmHID-R primer, 25 μL of 2×Phanta Max Master Mix, and 13 μL of ddH2O. Then, follow the PCR amplification program: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 55℃ for 15 s (the temperature can be adjusted according to different situations), 72℃ for 1 min (the time can be adjusted according to different situations), for 30 amplification cycles; finally, extend at 72℃ for 5 min.

[0103] After recovering the above DNA fragments, multiple fragments were ligated using the homologous recombination method with Novizan's ClonExpress Ultra One Step Cloning Kit V2. The ligation system was as follows: 1 μL of linearized vector (the amount can be adjusted according to the DNA concentration), 1 μL each of the target gene fragments (the amount can be adjusted according to the DNA concentration), 5 μL of 2×CE Mix, and ddH2O to a final volume of 10 μL. After mixing, the mixture was incubated at 50°C for 15 min, then cooled to 4°C or immediately placed on ice to obtain the corresponding ligation products.

[0104] The ligation product was then mixed with *E. coli* DH5α competent cells, incubated on ice for 30 min, followed by heat shock at 42°C for 1 min, and immediately placed on ice. After 2 min, 500 μL of LB medium was added, and the cells were incubated at 37°C for 1 h at 200 rpm. The cells were collected at the bottom of the tube by centrifugation at 5000 rpm for 1 min, resuspended in 100 μL of LB medium, and evenly spread onto plates containing antibiotics. The cells were incubated overnight at 37°C. Colony PCR identification confirmed the corresponding positive strain. Plasmids were extracted using the HiPure Plasmid MiniPrep Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.) and sequenced, yielding the correctly sequenced plasmid MGV3: pEAQ-GmCHIB1-P2A-AtPAL2-T2A-GmHID.

[0105] (3) Based on the above single expression cassette multi-gene expression vector, construct the multi-gene expression vector MGV4 containing two gene expression cassettes to express four genes: pEAQ-35S promoter-GmCHS8-P2A-GmCHR5-NOS teminator-35Spromoter-GmIFS1-P2A-AtMYB60-NOS teminator.

[0106] Construction method of MGV4: The multi-gene expression vector (single expression cassette) MGV1 was digested with NEB's Asc I restriction endonuclease; using the multi-gene expression vector (single expression cassette) MGV2 as a template, the full-length expression cassette of this vector from the 35S promoter to the NOS terminator was amplified by PCR using primers 39-40 and Novizan's 2×Phanta Max Master Mix, and the amplification reaction system was the same as above; after recovering the above DNA fragments, single fragments were ligated using Novizan's ClonExpressUltraOneStepCloningKit V2 via homologous recombination. Ligation, transformation and plasmid identification were the same as above to obtain plasmid MGV4.

[0107] (4) Based on the above single and two expression cassettes, a multi-gene expression vector MGV containing three gene expression cassettes expressing seven genes was constructed: pEAQ-35S promoter-GmCHIB1-P2A-AtPAL2-T2A-GmHID-NOS terminator-35S promoter-GmCHS8-P2A-GmCHR5-NOS teminator-35Spromoter-GmIFS1-P2A-AtMYB60-NOS teminator.

[0108] The multi-gene expression vector (two expression cassettes) MGV4 was digested with NEB's Pac I restriction endonuclease. Using the multi-gene expression vector (single expression cassette) MGV3 as a template, the full-length expression cassette from the 35S promoter to the NOS terminator was amplified by PCR using primers 41-42 and Novizan's 2×PhantaMax Master Mix. The amplification reaction system was the same as above. After recovering the above DNA fragments, single fragments were ligated using Novizan's ClonExpress Ultra One StepCloning Kit V2 via homologous recombination. The ligation, transformation, and plasmid identification were the same as above, and the final plasmid MGV was obtained.

[0109] 4. Construction of the synthetic pathway for direct precursors of daidzein synthesis

[0110] Based on the hypothesized synthetic pathways of the direct precursors of daidzein, glyceollidin I (GD1) and glyceollidin II (GD2) (… Figure 1 Using the multigene vector MGV (synthetic daidzein) constructed in step 3, the gene combinations MGV+GmI2'H, MGV+GmI2'H+GmIFR, MGV+GmI2'H+GmIFR+GmVR, MGV+GmI2'H+GmIFR+GmVR+GmPTS1, MGV+GmI2'H+GmIFR+GmVR+GmPTS1+GmD6aH, MGV+GmI2'H+GmIFR+GmVR+GmPTS1+GmD6aH+GmG4DT, and MGV+GmI2'H+GmIFR+GmVR+GmPTS1+GmD6aH+GmG2DT were co-expressed in tobacco leaves in a pyramid-shaped sequence.

[0111] Taking the gene combination MGV+GmI2'H co-expressed in tobacco leaves as an example, the process of Agrobacterium transformation and infection of tobacco leaves is as follows. Plasmids MGV and pEAQ-GmI2'H were transformed into Agrobacterium LBA4404 competent cells, respectively. The transformation method was as follows: 0.01-1 μg of plasmid DNA was added to each 100 μL of competent cells, and the mixture was stirred by hand at the bottom of the tube. The cells were then incubated sequentially on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and on ice for 5 minutes. 500 μL of LB liquid medium was added, and the cells were incubated at 28°C with shaking for 2-3 hours. The cells were collected by centrifugation at 6000 rpm for 1 minute. Approximately 100 μL of the supernatant was collected, gently resuspended, and spread onto LB agar plates containing kanamycin and rifampin. The plates were then incubated upside down at 28°C for 2-3 days. After colony PCR identification, the corresponding positive strains were determined. For cryopreservation, 50% glycerol was mixed with the bacterial culture at a 1:1 volume ratio and stored at -80°C.

[0112] Positive Agrobacterium bacteria were streaked onto LB solid medium containing kanamycin (50 mg / L) and rifampin (50 mg / L) for activation and cultured at 28°C for 2 days. Approximately pea-sized bacterial cells were inoculated into 5 mL of LB liquid medium containing kanamycin (50 mg / L) and rifampin (50 mg / L) and cultured at 28°C and 220 rpm for approximately 16 hours. When the bacterial culture showed an OD value... 600When the OD value reaches approximately 1.0, the bacterial culture is centrifuged at 4000 rpm for 8 min, the supernatant is discarded, and the bacterial cells are collected. The bacterial cells are resuspended in 500 μL of Agrobacterium permeation buffer (which contains 10 mM / L MgCl2, 10 mM / L morpholine ethanesulfonic acid, and 0.15 mM / L acetylsuccinone), and the final OD value of each bacterial cell is adjusted. 600 With a value of 0.2, different bacteria were mixed and allowed to stand at room temperature in the dark for 2-3 hours before being injected into tobacco leaves. The bacterial solution was slowly injected into the underside of the tobacco leaves using a 1 mL needleless syringe. The tobacco was kept in the dark overnight after injection and then cultured under the same conditions. Five days after injection, the tobacco leaves were cut off and dried in a benchtop freeze dryer for 2 days. The compounds in the tobacco leaves were then extracted directly or stored in a -80°C freezer for subsequent testing.

[0113] Finally, based on the Bench's tobacco platform for the efficient synthesis of isoflavone precursors, a synthetic pathway for the direct precursor of daidzein synthesis was reconstructed using an enzyme combination that produces daidzein. The consumption of the upstream precursor daidzein and the generation of new products were detected by liquid chromatography-mass spectrometry (LC-MS). The LC detection results of compounds extracted from tobacco leaves are as follows: Figure 2 As shown, daidzein was consumed after expressing GmI2'H; co-expression of GmVR and GmPTS1 produced a new peak that may be DHP, and the mass spectrometry data are shown in [link to mass spectrometry data]. Figure 3 A; then GmD6aH, GmD6aH and GmG4DT, GmD6aH and GmG2DT were added, and finally two pairs of new peaks that may be GD1 and GD2 were detected (i.e. GD1-3-gly and GD2-3-gly, GD1(1) and GD2(2) in the figure). The mass spectrometry data are shown in Figure 3 B, 3C.

[0114] 5. Isolation and purification of compounds from tobacco leaves

[0115] Leaf samples from *Nicotiana benthamiana* were injected with a mixed bacterial culture of approximately 400 strains that may have synthesized GD1 and GD2 compounds. The injected culture was then dried and pulverized in a freeze dryer to obtain approximately 150 g of leaf powder. The powder was extracted four times with 12 L of methanol under ultrasonic assistance. After each extraction, solid impurities were filtered off, and the solvent in the filtrate was removed using a rotary evaporator to obtain the total product extract. The total product extract was mixed with 50 g of silica gel and subjected to coarse separation using a silica gel column. A stepwise gradient elution was performed using petroleum ether-ethyl acetate (from 9:1 to 0:1), ethyl acetate-methanol (1:1), and 100% methanol, collecting 11 polar fractions. The fractions were analyzed by LC-MS, and fractions containing the target compounds were combined and the solvent was removed using a rotary evaporator. The concentrated fractions were then subjected to a gradient elution using a 26 mm × 206 mm C18 column with acetonitrile (the ratio was adjusted according to the polarity of the fraction). The fractions were collected and analyzed by LC-MS. Fractions containing the same target compounds were combined and dried. Impure fractions were further separated using a 13 mm × 300 mm silica gel column, eluted with acetonitrile (the ratio adjusted according to the polarity of the fraction), and the fractions were collected and analyzed by LC-MS. Fractions containing the same target compound were combined and dried. Fractions still impure were purified using semi-preparative liquid chromatography (HPLC), eluted with acetonitrile (the ratio adjusted according to the polarity of the fraction), and the fractions with a distinct absorption peak at 280 nm were collected. Fractions containing the target compound were identified by LC-MS, and then the fractions were combined and structurally identified by nuclear magnetic resonance spectroscopy (NMR).

[0116] After analyzing the potential direct precursors to daidzein synthesis, GD1 and GD2 (i.e., ...) were found to be synthesized. Figure 2 Enrichment, separation, purification, and NMR analysis of tobacco leaves containing GD1-3-gly and GD2-3-gly (GD1 and GD2) successfully identified a pair of novel compounds with the highest retention times: glycosylated GD1 and GD2 (i.e., GD1 and GD2). Figure 2 The NMR spectra of GD1-3-gly and GD2-3-gly are shown in [reference needed]. Figures 4-7 Therefore, it is speculated that the other pair of new peaks are unglycosylated GD1 and GD2 (i.e., Figure 2 GD1(1) and GD2(2) in the middle.

[0117] GD1-3-gly and GD2-3-gly were hydrolyzed using β-glucosidase. The specific procedure was as follows: 0.1 mg each of the purified GD1-3-gly and GD2-3-gly were weighed and added to 100 μL of 100 mg / mL β-glucosidase (purchased from Yuanye Biotechnology, 100 u / g) aqueous solution. The mixture was incubated at 65 °C for 30 min, then extracted twice with 200 μL of ethyl acetate. The ethyl acetate was dried under nitrogen, and then redissolved in 100 μL of methanol. After centrifugation at 12000 rpm, the mixture was analyzed by liquid chromatography using the same method as above. The results showed that the peaks of unglycosylated GD1 and GD2 were detectable. Figure 8 As shown, GD1 and GD2 have been successfully synthesized in Nicotiana benthamiana, laying the foundation for subsequent screening of daidzein synthases.

[0118] Example 2: Screening and functional identification of candidate daidzein synthases

[0119] Based on the reconstructed pathway for direct precursor synthesis of daidzein in Example 1, 12 candidate daidzein synthase genes were screened using bioinformatics co-expression analysis. These genes were highly co-expressed with the known genes involved in the conversion of daidzein to GD1 and GD2 and were induced by Phytophthora soybeanae. The genes were cloned and expression vectors were constructed; primers and gene information are shown in Tables 1 and 2, respectively.

[0120] 1. Bioinformatics analysis implementation process:

[0121] (1) Co-expression analysis: Using the validated downstream genes for daidzein synthesis, GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, GmG4DT, and GmG2DT, as well as the key isoflavone synthase GmIFS1, as "bait," co-expression gene information was obtained from the Glycine max RNA-seq database (https: / / plantrnadb.com / soybean / ). The top 20 co-expressed genes for each "bait" were obtained. First, overlapping genes were screened to narrow down the pool. Then, gene expression patterns under different induction conditions, WGCNA correlations, and functional annotations were analyzed to further screen for co-expression genes. Finally, 12 candidate genes encoding daidzein synthase, GmCYP71D8a, GmCYP71D8b, GmCYP82A4a, GmCYP82A2a, and PGS1-8 (corresponding GenBank accession numbers: NM_0013174, respectively), were selected. Functional characterization was performed on 83.2, XM_003537409.4, NM_001317501.1, XM_003546576.4, NM_001254191.2, XM_003548157.5, NM_001255341.3, NM_001254151.3, XM_003537846.4, NM_001251012.2, XM_003538926.5 and NM_001255757.3.

[0122] (2) Functional verification of candidate daidzein synthase genes: The function of candidate daidzein synthase genes was verified using Nicotiana benthamiana. The above 12 candidate genes encoding daidzein synthase were cloned and pEAQ-HT tobacco expression vectors were constructed. The cloning and construction methods are described in Example 1. The primers used and the gene information of the clones are shown in Tables 1 and 2, respectively. Each candidate daidzein synthase gene was co-expressed in Nicotiana benthamiana with the gene combinations MGV+GmI2'H+GmIFR+GmVR+GmPTS1+GmD6aH+GmG4DT and MGV+GmI2'H+GmIFR+GmVR+GmPTS1+GmD6aH+GmG2DT to screen for functional daidzein synthases. The Agrobacterium infection method was the same as in Example 1.

[0123] See the screening results Figure 9This indicates that four enzymes (GmCYP71D8a, GmCYP71D8b, GmCYP82A4a, and GmCYP82A2a) can further catalyze the conversion of daidzein direct precursors GD1(1) (marked 1 in the figure) and GD2(2) (marked 2 in the figure) into new compounds. Among them, GmCYP71D8a and GmCYP71D8b can both catalyze the synthesis of two new compounds from GD1(1), which were identified structurally as daidzein 1-9-glucose (Glyceollin I-9-glycoside (marked 3 in the figure) and daidzein 1 (marked 4 in the figure), GmCYP71D8a and GmCYP71D8b were renamed GmGS1a and GmGS1b, respectively; GmCYP82A4a can catalyze the conversion of GD2(2) to form two new compounds, which were identified by structure as daidzein 2-9-glucose (marked 5 in the figure) and daidzein 2 (marked 6 in the figure). GmCYP82A4a was renamed GmGS2a; GmCYP82A2a can catalyze GD2(2) to produce three new compounds, which were identified by separation and characterization as glycerofuran (marked 7 in the figure), 13-epi-glyceollin 3 (marked 8 in the figure) and a new glyceollin structure. The new glyceollin structure was named glyceollin 7 (marked 9 in the figure), and GmCYP82A2a was renamed GmGS3e / 7.

[0124] (3) Homologous gene screening: Genes were searched in the soybean genome using the BLAST function in NCBI, sequence alignment was performed using MEGA11 software, and phylogenetic tree analysis was conducted using the neighbor-joining method. Results are shown below. Figure 10 .

[0125] By testing the function of other GmCYP82A homologs in the soybean genome, two functional GmCYP82A homologs that can act on GD2(2) were further identified, namely GmCYP82A4b and GmCYP82A2b. The results are shown in […]. Figure 9As can be seen from the figure, GmCYP82A4b has the same catalytic function as GmCYP82A4a, hence it is named GmGS2b; while GmCYP82A2b exhibits a different function from GmCYP82A2a, catalyzing the synthesis of new compounds daidzein 3-9-glucose (marked 10 in the figure) and daidzein 3 (marked 11 in the figure) from GD2(2), therefore, GmCYP82A2b is named GmGS3. The final pathway of daidzein biosynthesis was finally identified as follows: Figure 11 As shown.

[0126] In summary, six daidzein synthases were successfully identified, and daidzein 1, daidzein 2, daidzein 3, daidzein 1-9-glucose, daidzein 2-9-glucose, daidzein 3-9-glucose, 13-epi-daidzein 3, glycerofuran, and a novel daidzein, namely daidzein 7, were synthesized. Secondary mass spectrometry data for all synthesized products are shown below. Figure 12 As shown, the NMR spectra of daidzein 1-9-glucose, daidzein 1, daidzein 2-9-glucose, daidzein 2, glycerol furan, 13-epi-daidzein 3, daidzein 7, daidzein 3-9-glucose, and daidzein 3 are as follows: Figure 13 , 15 As shown in Figures 17, 19, 21, 23, 25, 27, and 29, the corresponding NMR data are as follows: Figure 14 , 16 As shown in 18, 20, 22, 24, 26, 28, 30.

[0127] This invention provides a method and approach for the biosynthesis of daidzein precursors and daidzein based on daidzein catalysis. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A biosynthetic method for synthesizing daidzein precursors based on daidzein catalysis, characterized in that, Starting with Nicotiana benthamiana, a daidzein precursor was obtained by co-expressing genes for daidzein synthesis and downstream genes for daidzein synthesis in Nicotiana benthamiana mediated by Agrobacterium tumefaciens. The downstream genes for daidzein synthesis are GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, and GmG4DT, or GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, and GmG2DT.

2. The biosynthesis method according to claim 1, characterized in that, The gene for synthesizing daidzein consists of seven genes: phenylalanine ammonia-lyase gene AtPAL2, chalcone synthase gene GmCHS8, chalcone reductase gene GmCHR5, chalcone isomerase gene GmCHI1B1, isoflavone synthase gene GmIFS1, 2-hydroxyisoflavone dehydratase gene GmHID, and transcription factor AtMYB60.

3. The biosynthesis method according to claim 2, characterized in that, The gene for synthesizing daidzein is expressed via plasmid MGV; wherein, the plasmid MGV is a multi-gene expression vector containing three gene expression cassettes to express the seven genes: pEAQ-35S promoter-GmCHIB1-P2A-AtPAL2-T2A-GmHID-NOS terminator-35S promoter-GmCHS8-P2A-GmCHR5-NOS teminator-35S promoter-GmIFS1-P2A-AtMYB60-NOSteminator.

4. The biosynthesis method according to claim 2, characterized in that, The downstream gene for daidzein synthesis is expressed via the expression vector pEAQ-HT, which contains the downstream gene for daidzein synthesis.

5. A biosynthetic method for the synthesis of daidzein based on daidzein catalysis, characterized in that, Starting with Nicotiana benthamiana, daidzein was synthesized by co-expressing genes for daidzein synthesis, downstream genes for daidzein synthesis, and daidzein synthase genes in Nicotiana benthamiana mediated by Agrobacterium.

6. The biosynthesis method according to claim 5, characterized in that, The gene for synthesizing daidzein is expressed via plasmid MGV; wherein, plasmid MGV is a multi-gene expression vector containing three gene expression cassettes expressing seven genes; The seven genes are phenylalanine ammonia-lyase gene AtPAL2, chalcone synthase gene GmCHS8, chalcone reductase gene GmCHR5, chalcone isomerase gene GmCHI1B1, isoflavone synthase gene GmIFS1, 2-hydroxyisoflavone dehydratase gene GmHID, and transcription factor AtMYB60.

7. The biosynthesis method according to claim 5, characterized in that, The daidzeins include daidzein 1, daidzein 2, daidzein 3, 13-epi-daidzein 3, and daidzein 7.

8. The biosynthesis method according to claim 7, characterized in that, in, The daidzein 7 is a novel daidzein with the following chemical structural formula:

9. The biosynthesis method according to claim 5, characterized in that, The downstream genes for daidzein synthesis are GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, GmG4DT, or GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, GmG2DT; the daidzein synthase genes include any one or a combination of several of GmGS1a, GmGS1b, GmGS2a, GmGS2b, GmGS3e / 7, and GmGS3.

10. The biosynthesis method according to claim 9, characterized in that, (1) When the downstream gene for daidzein synthesis is GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, or GmG4DT, and the daidzein synthase gene is GmGS1a or GmGS1b, the daidzein is daidzein 1. (2) When the downstream gene for daidzein synthesis is GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, or GmG2DT, and the daidzein synthase gene is GmGS2a or GmGS2b, the daidzein is daidzein 2. (3) When the downstream genes for daidzein synthesis are GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, and GmG2DT, and the daidzein synthase gene is GmGS3e / 7, the daidzein is 13-epi-daidzein3 and daidzein7. (4) When the downstream genes for daidzein synthesis are GmI2'H, GmIFR, GmVR, GmPTS1, GmD6aH, and GmG2DT, and the daidzein synthase gene is GmGS3, the daidzein is daidzein 3.

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