Method for constructing nicotiana benthamiana platform for synthesizing isoflavone compounds and application thereof

Abstract

This invention relates to the field of biotechnology, specifically to the construction and application of a Benzodiacetic tobacco chassis for synthesizing isoflavones. This invention reconstructs the upstream pathway for isoflavone synthesis in Benzodiacetic tobacco, further identifies and screens key rate-limiting enzymes for isoflavone synthesis in Benzodiacetic tobacco, and utilizes 2A peptides to co-express multiple key genes, further introducing flavonoid synthesis transcription factors to construct a Benzodiacetic tobacco chassis for synthesizing isoflavones. This Benzodiacetic tobacco chassis can efficiently synthesize daidzein and genistein. Furthermore, based on the constructed high-yield Benzodiacetic tobacco platform for daidzein and genistein, various isoflavones can be further synthesized, such as daidzein, genistein, puerarin, gentiopicrin, chickpea extract A, and stigmosiderin, paving the way for the industrialization of plant biosynthesis of various bioactive isoflavones and thus possessing broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for constructing and applying a Benzodiacetic tobacco chassis for synthesizing isoflavone compounds. Background Technology

[0002] Isoflavones are a class of flavonoids derived from the phenylpropanoid metabolic pathway, primarily synthesized in legumes. They play crucial roles not only in plant growth and development, responses to biotic and abiotic stresses, and the induction of root nodule formation in legumes, but also in human health, possessing significant health benefits and various potential pharmacological activities. Therefore, plant isoflavones have immense commercial value in agrochemicals, pharmaceuticals, health products, and cosmetics. However, their low abundance and structural complexity in plants severely hinder the acquisition of sufficient quantities of these highly active isoflavones through traditional plant extraction and chemical synthesis, thus limiting the large-scale production of isoflavone products using standardized industrial processes. Therefore, developing alternative sources of isoflavones is a pressing challenge. Although several studies have reported on the production of isoflavone compounds using microorganisms as a substrate, the normal expression of plant-derived enzymes and the synthesis of complex isoflavone compounds in microorganisms remain significant challenges. Furthermore, microbial industrial fermentation involves issues such as high costs, biosafety, and environmental pollution. Nicotiana benthamiana, as a non-crop model plant with high biomass and extensive secondary metabolic pathways, possesses a mature technological foundation for transient and stable transformation, enabling green and sustainable production. It is considered an excellent host for heterologous biosynthesis of plant natural products. Therefore, heterologous biosynthesis of isoflavones using Nicotiana benthamiana is an attractive green alternative, laying the scientific theoretical and technological foundation for the research on the medicinal functions of isoflavone compounds and their large-scale industrialization. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for constructing a universal chassis for synthesizing isoflavone compounds from Tobacco Benzovia, which addresses the various shortcomings of existing microbial biosynthesis platforms.

[0004] Another technical problem to be solved by the present invention is to provide the application of the Benedictine tobacco chassis constructed by the above construction method in the synthesis of various isoflavone compounds.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for constructing a Benzoinus chassis for synthesizing isoflavones involves co-expressing the encoding genes of phenylalanine ammonia-lyase PAL, chalcone synthase CHS, chalcone reductase CHR, chalcone isomerase CHI, isoflavone synthase IFS, 2-hydroxyisoflavone dehydratase HID, and transcription factors into Agrobacterium-mediated Benzoinus to construct the first Benzoinus chassis for synthesizing isoflavones.

[0007] Alternatively, by using an expression vector, the encoding genes of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), isoflavone synthase (IFS), and transcription factors can be co-expressed in Agrobacterium-mediated Nicotiana benthamiana to construct a second Nicotiana benthamiana chassis for synthesizing isoflavone compounds.

[0008] The gene encoding phenylalanine ammonia-lyase (PAL) includes any one of AtPAL1, AtPAL2, AtPAL3, GmPAL1.1, GmPAL2.1, GmPAL2.3, MtPAL, MsPAL, TpPAL, TrPAL, and NbPAL; the GenBank accession number or nucleotide sequence corresponding to the gene encoding PAL is as follows: AY079363.1, NM_115186.4, NM_120505.4, NM_001357056.1, NM_001250027.2, NM_001357054.1, XM_003590423.3, X58180.1, AB236800.1, SEQ ID NO.1 (GenBank accession number for amino acid sequence as shown in KAK2401244.1), MK689226.1.

[0009] The encoding gene for chalcone synthase CHS includes any one of GmCHS7, GmCHS8, MtCHS, MsCHS, TpCHS, TrCHS, and GuCHS; the GenBank accession number or nucleotide sequence corresponding to the encoding gene for chalcone synthase CHS is as follows: NM_001353380.1, AY237728.1, XM_039829904.1, U01018.1, XM_045955819.1, SEQ ID NO.2 (the GenBank accession number for the amino acid sequence is shown as KAK2425798.1), KY748118.1_.

[0010] The encoding gene for chalcone reductase CHR includes any one of GmCHR5, GmCHR1, GmCHR6, MtCHR, MsCHR, TpCHR, TrCHR, and GuCHR; the GenBank accession number or nucleotide sequence corresponding to the encoding gene for chalcone reductase CHR is as follows: LC309095.1, NM_001249044.2, NM_001367007.1, XM_003601522.4, X82368.1, XM_045935161.1, SEQ ID NO.3 (GenBank accession number for amino acid sequence as shown in KAK2389066.1), and D86559.1.

[0011] The encoding gene for the chalcone isomerase CHI includes any one of GmCHI1B1, GmCHI1A, GmCHI2, MtCHI, TpCHI, TrCHI, and NbCHI; the GenBank accession number or nucleotide sequence corresponding to the encoding gene for the chalcone isomerase CHI is as follows: NM_001249826.2, AY595413.1, NM_001249839.2, XM_003592715.3, XM_045963556.1, SEQ ID NO.5 (the GenBank accession number for the amino acid sequence is shown as KAK2405643.1), and SEQ ID NO.4.

[0012] The encoding genes for isoflavone synthase IFS include GmIFS1, MtIFS, and GuIFS; the GenBank accession numbers corresponding to the encoding genes for isoflavone synthase IFS are AF195798.1, MH450243.1, and JF912327.1, respectively.

[0013] The gene encoding the 2-hydroxyisoflavone dehydratase HID is GmHID, and the corresponding GenBank accession number is AB154415.1.

[0014] The transcription factors include any one or a combination of AtMYB12, GmMYB12B2 and AtMYB60; the GenBank accession numbers corresponding to the encoding genes of the transcription factors are NM_130314.4_, JF510467.1 and NM_100755.3, respectively.

[0015] Preferably, the gene encoding the phenylalanine ammonia-lyase PAL is AtPAL2; the gene encoding the chalcone synthase CHS is GmCHS8; the gene encoding the chalcone reductase CHR is GmCHR5; the gene encoding the chalcone isomerase CHI is GmCHI1B1; the gene encoding the isoflavone synthase IFS is GmIFS1; and the transcription factor is GmMYB12B2 and / or AtMYB60.

[0016] More preferably, when the constructed *Tobacco Bungei* chassis is a first *Tobacco Bungei* chassis, the transcription factor is AtMYB60; or, when the constructed *Tobacco Bungei* chassis is a second *Tobacco Bungei* chassis, the transcription factors are GmMYB12B2 and AtMYB60.

[0017] The coding genes are linked by a 2A peptide.

[0018] Specifically, the 2A peptide is either a P2A peptide or a T2A peptide.

[0019] Specifically, the amino acid sequence of the P2A peptide is shown in SEQ ID NO.7, and the corresponding nucleotide sequence is shown in SEQ ID NO.8; the amino acid sequence of the T2A peptide is shown in SEQ ID NO.9, and the corresponding nucleotide sequence is shown in SEQ ID NO.10.

[0020] The expression vector is pEAQ-HT; the co-expression into Agrobacterium-mediated Tobacco Benzoinus is specifically achieved by co-expressing the expression vector containing the coding gene into Agrobacterium, and then using Agrobacterium to infect Tobacco Benzoinus leaves.

[0021] Specifically, the expression vector containing the coding gene is a single-gene expression vector or a multi-gene expression vector containing multiple expression cassettes.

[0022] Specifically, when the constructed *Nicotiana benthamiana* chassis is the first *Nicotiana benthamiana* chassis, the single-gene expression vectors are pEAQ-AtPAL2, pEAQ-GmCHS8, pEAQ-GmCHR5, pEAQ-GmCHI1B1, pEAQ-GmIFS1, pEAQ-GmHID, and pEAQ-AtMYB60; the multi-gene expression vector containing multiple expression cassettes is the multi-gene expression vector MGV8 containing three expression cassettes: pEAQ-35S promoter-GmCHS8-P2A-GmCHR5-NOS teminator-35S promoter-GmIFS1-P2A-AtMYB60-NOS teminator-35S promoter-GmCHIB1-P2A-AtPAL2-T2A-GmHID-NOSterminator;

[0023] Alternatively, when the constructed *Nicotiana benthamiana* chassis is a second *Nicotiana benthamiana* chassis, the single-gene expression vector is pEAQ-AtPAL2, pEAQ-GmCHS8, pEAQ-GmIFS1, pEAQ-GmMYB12B2, and pEAQ-AtMYB60; the multi-gene expression vector containing multiple expression cassettes is the multi-gene expression vector MGV10 containing two expression cassettes: pEAQ-35S promoter-GmCHS8-P2A-GmMYB12B2-T2A-AtPAL2-NOS teminator-35S promoter-GmIFS1-P2A-AtMYB60-NOS teminator.

[0024] The application of the Benedictine chassis constructed by the above method in the synthesis of isoflavone compounds is also within the scope of protection of this invention.

[0025] The isoflavone compounds include any one or a combination of several of the following: daidzein, genistein, daidzein, genistein, puerarin, methylated isoflavone gentiopicrin, chickpea ginsenoside A, and mediopterygium oleracea.

[0026] Specifically, the daidzein, genistein, and puerarin are glycosylated isoflavones; the styrosinin, chickpea extract A, and medroxylin are methylated isoflavones.

[0027] In some embodiments of the present invention

[0028] (i) Synthesizing daidzein using the first Benedictine chassis;

[0029] or,

[0030] (ii) Synthesize lignin dyes using the second Benedict's tobacco chassis;

[0031] or,

[0032] (iii) Using the first Benedictine chassis as the starting chassis, daidzein was synthesized by overexpressing glycosyltransferase GmUGT4.

[0033] or,

[0034] (iiii) Using the second Nicotiana benthamian carcass as the starting carcass, genistein was synthesized by overexpressing the glycosyltransferase GmUGT4;

[0035] or,

[0036] (iv) Using the first Nicotiana benthamian carcass as the starting carcass, puerarin was synthesized by overexpressing the glycosyltransferase PlUGT43.

[0037] or,

[0038] (v) Using the first Benedictine chassis as the starting chassis, methylated isoflavone argentin was synthesized by overexpressing 2'-hydroxyisoflavone-4'-O-methyltransferase MtHI4'OMT.

[0039] or,

[0040] (vi) Using the second Benedictine chassis as the starting chassis, 2'-hydroxyisoflavone-4'-O-methyltransferase MtHI4'OMT was overexpressed to synthesize chickpea protein A;

[0041] or,

[0042] (vii) Using the first Benedictine chassis as the starting chassis, 2'-hydroxyisoflavone-4'-O-methyltransferase MtHI4'OMT, isoflavone 2'-hydroxylase MtI2'H, isoflavone reductase MsIFR, vestitone reductase MsVR and santalin synthase MtPTS were overexpressed to synthesize santalin.

[0043] Beneficial effects:

[0044] (1) In view of the various shortcomings of the existing microbial biosynthesis platform, this invention provides a method for constructing a Benzodiacetic chassis for synthesizing isoflavone compounds by co-expressing the encoding genes of related genes of this pathway, such as phenylalanine ammonia-lyase PAL, chalcone synthase CHS, chalcone reductase CHR, chalcone isomerase CHI, isoflavone synthase IFS, 2-hydroxyisoflavone dehydratase HID and transcription factors, into Agrobacterium-mediated Benzodiacetic tobacco using an expression vector.

[0045] (2) The method for constructing the Benzodiacetic tobacco chassis for synthesizing isoflavones described in this invention can achieve high yields of daidzein and genistein. The yield of daidzein per kilogram of dry Benzodiacetic tobacco reaches 4.28 g, and the yield of genistein reaches 10.26 g. Compared with the unoptimized version, the yield of daidzein is increased by approximately 6.8 times, and the yield of genistein is increased by approximately 8.5 times. These are the highest yields to date for plant chassis synthesis, while the highest yields to date for microbial chassis are 85.4 mg / L and 76.37 mg / L, respectively.

[0046] (3) Based on the established high-yield daidzein and genistein platform in *Tobacco Benzoinus*, this invention further synthesized three glycosylated isoflavones, including daidzein, genistein, and puerarin, as well as three methylated isoflavones, including gentiopicrin, chickpea extract A, and stigmosiderin. Yields were achieved at 18.74 g of daidzein, 5.36 g of genistein, 4.18 g of puerarin, 2.26 g of gentiopicrin, 4.08 g of chickpea extract A, and 0.72 g of stigmosiderin per kilogram of dry weight of *Tobacco Benzoinus*. Among these, daidzein, genistein, and puerarin were obtained in a microbial chassis at yields of 73.2 mg / L, 202.7 mg / L, and 72.8 mg / L, respectively, while gentiopicrin, chickpea extract A, and stigmosiderin were synthesized heterologously and de novo for the first time.

[0047] (4) This invention constructs a universal tobacco chassis suitable for the efficient synthesis of different types of isoflavones and realizes the heterologous synthesis of a variety of active isoflavone compounds, paving the way for the industrialization of plant biosynthesis of various active isoflavones. Attached Figure Description

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0049] Figure 1 This represents the upstream biosynthetic pathway of plant isoflavones.

[0050] Figure 2 Standard curves for the quantification of isoflavone upstream biosynthetic products naringenin, glycyrrhizin, daidzein and genistein.

[0051] Figure 3 This study identifies the rate-limiting enzymes in the upstream biosynthetic pathway of isoflavones. A represents the reconstruction of the upstream biosynthetic pathway of isoflavones; B represents the effects of CHI and HID on the synthesis of daidzein and genistein; and C represents the identification of the rate-limiting enzymes in the common pathway of phenylpropanone synthesis. In this study, GC1 represents the gene combination GmPAL2.1+GmC4H+Gm4CL2+GmCHS7; GC2 represents the gene combination GmCHS7+GmCHR5; nd indicates not detected; DW represents dry weight; ** indicates p<0.01, **** indicates p<0.0001.

[0052] Figure 4This study aimed to screen for highly efficient isoenzymes. In the diagram, A represents different combinations of PAL and GmCHS7 expression; B (left) represents different combinations of CHS and AtPAL2 expression, and (right) represents different combinations of CHS and AtPAL2+GmCHR5 expression; C represents different combinations of CHR and AtPAL2+GmCHS8 expression; D represents different combinations of CHI and AtPAL2+GmCHS8+GmCHR5+GmIFS1+GmHID expression; E (left) represents different combinations of IFS and AtPAL2+GmCHS8+GmCHR5+GmCHI1B1+GmHID expression, and (right) represents different combinations of IFS and AtPAL2+GmCHS8 expression. Relative yield = yield / highest yield in this comparison group.

[0053] Figure 5 Screening for multi-gene combinations linked by 2A peptides. Where A represents gene combinations linked by 2A peptides; B represents comparisons of daidzein yield using multi-gene expression vectors. MGV indicates a multi-gene vector; DW indicates dry weight; ** indicates p < 0.01; GC3 represents the gene combination AtPAL2+GmCHR5+GmCHS8+GmCHI1B1+GmIFS1+GmHID; GC4 represents the gene combination GmCHR5+GmCHI1B1+GmIFS1+GmHID; GC5 represents the gene combination AtPAL2+GmCHI1B1+GmIFS1+GmHID; GC6 represents the gene combination GmCHI1B1+GmIFS1+GmHID; and GC7 represents the gene combination AtPAL2+GmIFS1+GmHID.

[0054] Figure 6 Screening of transcription factors. Figure A shows the screening of positive regulatory factors; Figure B shows the screening of anthocyanin repressor factors; Figure C shows the effect of single or combined transcription factors on daidzein synthesis; Figure D shows the effect of single or combined transcription factors on genistein synthesis. In the figure, GC8 represents the gene combination AtPAL2+MGV2+GmCHI1B1+GmIFS1+GmHID; GC9 represents the gene combination AtPAL2+GmCHS8+GmIFS1; DW represents dry weight; ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0055] Figure 7This diagram illustrates the assembly of genes for efficient isoflavone synthesis. Figure A shows a schematic diagram of a multi-expression cassette vector used for daidzein (left) and genistein (right) synthesis; Figure B compares daidzein yield under different vectors; Figure C compares genistein yield under different vectors. In the diagram, GC10 represents the gene combination AtPAL2+MGV2+GmCHI1B1+GmIFS1+GmHID+AtMYB60; GC11 represents the gene combination AtPAL2+GmCHS8+GmIFS1+GmMYB12B2+AtMYB60; DW represents dry weight; * indicates p<0.05.

[0056] Figure 8 This is a downstream biosynthetic pathway for isoflavones.

[0057] Figure 9 Standard curves for the quantification of daidzein, genistein, puerarin, styracin, chickpea extract A, and stigmosiderin, products of the downstream biosynthetic pathway of isoflavones.

[0058] Figure 10 This section describes the synthesis of glycosylated isoflavones. In the figures, A represents the liquid chromatography results of daidzein, genistein, and puerarin; B represents the yield of daidzein; C represents the yield of genistein; and D represents the yield of puerarin. EIC represents the extract ion chromatogram; and DW represents the dry weight.

[0059] Figure 11 This section describes the synthesis of methylated isoflavones. In the figures, A represents the liquid chromatography results of genistein and chitosan A; B represents the yield of genistein; and C represents the yield of chitosan A. EIC indicates the extracted ion chromatogram; DW represents dry weight; and nd indicates not detected.

[0060] Figure 12 This diagram represents the synthesis of santalin. A shows the reconstruction of the santalin synthesis pathway; B shows the santalin yield. EIC represents the extracted ion chromatogram; DW represents dry weight; nd indicates not detected. GC12 represents MtHI4'OMT+MtI2'H+MsIFR+MsVR+MtPTS. Detailed Implementation

[0061] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0062] 1. In the following embodiments, the experimental materials and reagents are as follows:

[0063] (1) Source of materials:

[0064] Plant materials: Seeds of Nicotiana benthamiana and Arabidopsis thaliana (Columbia type) were preserved in our laboratory; soybean seeds were from the soybean variety William 82 from Nanjing Agricultural University; alfalfa seeds were from the variety A17 from the University of Science and Technology of China; alfalfa and licorice seeds were purchased from the Medicinal Herb Cultivation Guide website; red clover, white clover and snapdragon plants were purchased from the Euonymus alatus base of Xidu Botanical Garden; and strawberry plants were purchased from Huadu Horticulture.

[0065] Vector: The tobacco expression empty vector pEAQ-HT was preserved in our laboratory.

[0066] 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.

[0067] Standards: All compound standards were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0068] (2) Solution formulation

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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 strain preservation.

[0073] Agrobacterium permeate: 10 mM / L MgCl2, 10 mM / L morpholine ethanesulfonic acid and 0.15 mM / L acetylsyl syringone, used for transient conversion of tobacco.

[0074] Hydrochloric acid solution: 1 mol / L hydrochloric acid, used to hydrolyze the glycosyl compounds in the sample.

[0075] 2. In the following examples, the primers and gene information used are shown in Table 1 and Table 2, respectively.

[0076] Table 1 Primers and their sequences used in this invention

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] Table 2 Gene information used in this invention

[0085]

[0086]

[0087]

[0088] 3. In the following examples, the specific experimental procedures for the extraction and detection of compounds from tobacco leaves are as follows:

[0089] (1) Routine sample preparation: Weigh 10 mg dry weight (DW) of tobacco leaves, 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 vial for sample loading.

[0090] (2) Preparation of acid-hydrolyzed deglycosylated samples: Weigh 10 mg of lyophilized tobacco leaves, dissolve in 400 μL of 80% methanol, sonicate at room temperature for 45 min, and centrifuge at 12000 rpm for 10 min. Take 250 μL of supernatant into a 2 mL centrifuge tube, add 750 μL of 1 mol / L hydrochloric acid solution, and incubate at 95℃ for 2 h. After cooling, extract twice with 1 mL of ethyl acetate, take the supernatant, blow dry, dissolve thoroughly in an appropriate amount of 80% methanol, centrifuge, and take the supernatant for sample loading. This method is used for the quantitative determination of the yield of naringenin, glycyrrhizin, daidzein, genistein, gentiopicrin, and chickpea extract A.

[0091] (3) 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.

[0092] (4) 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.

[0093] (5) Preparation of standard curve: Weigh an appropriate amount of compound standard, add 80% methanol to prepare a 1 mg / mL standard stock solution, and dilute it sequentially to 200, 125, 100, 50, 25, 10, 4, 2, 1 and 0.5 mg / L standard solutions. Measure the peak area corresponding to different concentrations using high performance liquid chromatography / mass spectrometry to plot the standard curve for compound quantification.

[0094] 4. In the following examples, the constructed multi-gene expression vectors MGV1-10 are shown in Table 3.

[0095] Table 3 List of multi-gene expression vectors constructed in this invention

[0096]

[0097] 5. In the following examples, the transcription factor combination AmROS1 / AmDEL is described in: [1] Fatihah, HNNetal. The ROSEA1 and DELILA transcription factors control anthocyanin biosynthesis in Nicotiana benthamiana and Lilium flowers[J].; the transcription factor AtMYB12 is described in: [2] Pandey, A. et al. Co-expression of Arabidopsis transcription factor, AtMYB12, and soybean isoflavone synthase, GmIFS1, genes in tobacco leads to enhanced biosynthesis of isoflavones and flavonols resulting inosteoprotective activity[J].; the transcription factor GmMYB12B2 is described in: [3] Li, XWet al. AR2R3-MYB transcription factor, GmMYB12B2, affects the expression levels of flavonoid biosynthesis genes encoding key enzymes in transgenic Arabidopsisplants[J].; The transcription factor GmMYB176 is described in: [4] Yi, J. et al. A single-repeat MYB transcription factor, GmMYB176, regulates CHS8 gene expression and affects isoflavoneoid biosynthesis in soybean[J].; The transcription factor AtMYB60 is described in: [5] Park, J. Set al. Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as atranscriptional repressor of anthocyanin biosynthesis in lettuce (Lactucasativa)[J].; The transcription factor AtMYBL2 is described in: [6] Matsui, K., Umemura, Y. & Ohme-Takagi, M. AtMYBL2, a protein with a single MYB domain, acts as a negative regulator ofanthocyanin biosynthesis in Arabidopsis[J].; The transcription factor AtCPC is recorded in: [7] Zhu, HF, Fitzsimmons, K., Khandelwal, A. & Kranz, RGCPC, a single-repeat R3 MYB, is a negative regulator of anthocyanin biosynthesis in Arabidopsis[J].

[0098] Example 1: Identification of the key rate-limiting enzyme for heterologous synthesis of isoflavones in *Nicotiana benthamiana*

[0099] To identify the key rate-limiting enzymes for isoflavone synthesis in Nicotiana benthamiana, relevant genes from soybean (Glycine max L) were first cloned, including the gene GmPAL2.1 encoding phenylalanine ammonia-lyase (PAL), the gene GmC4H encoding cinnamic acid-4-hydroxylase (C4H), the gene Gm4CL2 encoding 4-coumaric acid-coenzyme-A-ligase (4CL), the gene GmCHS7 encoding chalcone synthase (CHS), the gene GmCHR5 encoding chalcone reductase (CHR), the gene GmCHI1B1 encoding chalcone isomerase (CHI), the gene GmIFS1 encoding isoflavone synthase (IFS), and the gene GmHID encoding 2-hydroxyisoflavone dehydratase (HID). The primer information is shown in Table 1, and the relevant gene information is shown in Table 2.

[0100] (1) Gene cloning: 100 mg of different tissues such as roots, stems and leaves of soybean were taken respectively, and plant RNA was extracted using a rapid universal plant RNA extraction kit (purchased from Huayueyang Biotechnology Co., Ltd.), and then reverse transcription kit was used to extract the plant RNA. One-Step gDNA Removal and cDNA Synthesis SuperMix (purchased from Beijing TransGen Biotech Co., Ltd.) was used to reverse transcribe cDNA. This cDNA was then used as a template for PCR amplification. The target gene was cloned using high-fidelity DNA polymerase 2x Phanta MaxMaster Mix (purchased from Nanjing Novizan Biotechnology Co., Ltd.) and corresponding upstream and downstream primers. After agarose gel electrophoresis, the PCR amplification products were recovered and purified using a Quick Gel Extraction Kit (purchased from Beijing TransGen Biotech Co., Ltd.) to obtain the corresponding target gene fragment. The reverse transcription reaction system and procedure, and the PCR amplification reaction system and procedure are as follows:

[0101] The reverse transcription reaction system was as follows: 50 ng - 5 μg RNA, 1 μL Oligo(dT), 10 μL 2X TS Reaction Mix, 1 μL RT / RI Enzyme Mix, 1 μL gDNA Remover, and 20 μL RNase-free Water. The reverse transcription program was: 42℃ for 30 min, then 85℃ for 5 min. After completion, the mixture was stored at -20℃.

[0102] The PCR amplification reaction system is as follows (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. The PCR amplification program is 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 35 amplification cycles; finally, 72℃ extension for 5 min.

[0103] (2) Construction of single-gene vectors: 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 reacted at 50℃ for 5 min, then cooled to 4℃ 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, heat-shocked in a 42°C water bath 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 a plate containing the resistant culture. The cells were then incubated overnight at 37°C. After colony PCR identification, the corresponding positive strains were determined. Plasmids were extracted using the HiPure Plasmid MiniPrep Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.) and sent for sequencing. The correctly sequenced plasmids were pEAQ-GmPAL2.1, pEAQ-GmC4H, pEAQ-Gm4CL2, pEAQ-GmCHS7, pEAQ-GmCHR5, pEAQ-GmCHI1B1, pEAQ-GmIFS1, and pEAQ-GmHID.

[0105] (3) Agrobacterium transformation: The pEAQ-GmPAL2.1, pEAQ-GmC4H, pEAQ-Gm4CL2, pEAQ-GmCHS7, pEAQ-GmCHR5, pEAQ-GmCHI1B1, pEAQ-GmIFS1 and pEAQ-GmHID plasmids constructed and correctly sequenced in step (2) 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. The cells were then incubated on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes. 500 μL of the plasmid DNA was then added. In LB liquid medium, culture with shaking at 28°C for 2–3 hours; collect bacteria by centrifugation at 6000 rpm for 1 minute, and resuspend approximately 100 μL of supernatant by gentle pipetting. Spread the bacterial block onto LB agar plates containing kanamycin and rifampin, and incubate upside down at 28°C for 2–3 days. After colony PCR identification, determine the corresponding positive strains. For cryopreservation, mix 50% glycerol with the bacterial suspension at a 1:1 volume ratio and store at -80°C.

[0106] (4) Agrobacterium infection of tobacco leaves: 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 OD... 600 When 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 permeate buffer, and the final OD value of each strain is adjusted. 600 The value is 0.2.

[0107] Based on the upstream biosynthetic pathway of plant isoflavones ( Figure 1Using a pyramid-shaped combination sequence, the gene combinations GmPAL2.1, GmPAL2.1+GmC4H, GmPAL2.1+GmC4H+Gm4CL2, GmPAL2.1+GmC4H+Gm4CL2+GmCHS7, GmPAL2.1+GmC4H+Gm4CL2+GmCHS7+GmCHR5, and GmPAL2 were co-expressed in tobacco leaves. The gene combinations are: 1+GmC4H+Gm4CL2+GmCHS7+GmCHR5+GmCHI1B1, GmPAL2.1+GmC4H+Gm4CL2+GmCHS7+GmCHR5+GmCHI1B1+GmIFS1, and GmPAL2.1+GmC4H+Gm4CL2+GmCHS7+GmCHR5+GmCHI1B1+GmIFS1+GmHID. Specifically, based on the above gene combinations, the bacterial suspensions of Agrobacterium strains containing the above genes are mixed and incubated at room temperature in the dark for 2-3 hours before being injected into tobacco leaves. The bacterial suspension is slowly injected into the underside of the tobacco leaves using a 1 mL needleless syringe. The tobacco is then kept in the dark overnight after injection and subsequently cultured under the same conditions. Five days after injection, the tobacco leaves are cut and dried in a benchtop freeze dryer for two days. The compounds are then extracted and detected directly, or stored at -80°C.

[0108] Standard curves for the quantitative analysis of isoflavone upstream biosynthetic products naringenin, glycyrrhizin, daidzein, and genistein are shown below. Figure 2 As shown in the figure. The experimental results of compound synthesis after sequential expression of the GmPAL2.1, GmC4H, Gm4CL2, GmCHS7, GmCHR5, GmCHI1B1, GmIFS1, and GmHID genes in tobacco leaves are as follows. Figure 3 As shown in A. The results showed that the expression of different gene combinations (GC) produced different intermediates of isoflavone synthesis: expression of only GmPAL2.1, GmC4H and Gm4CL2 could not detect the synthesis of any target compound, while the addition of GmCHS7 detected the accumulation of naringenin (1); the addition of GmCHR5 resulted in the accumulation of glycyrrhizin (2) and a decrease in the accumulation of naringenin (1), while the addition of GmCHI1B1 still only accumulated naringenin (1) and glycyrrhizin (2), but the addition of GmIFS1 detected the accumulation of daidzein (3) and genistein (4), and the accumulation of naringenin (1) and glycyrrhizin (2) was reduced. The addition of the last enzyme gene GmHID did not significantly affect the accumulation of compounds. Therefore, based on the sequence and yield of the detected compounds, CHS enzyme, CHR enzyme, and IFS enzyme were initially screened as three rate-limiting enzymes involved in the synthesis of isoflavones, namely naringenin (1), glycyrrhizin (2), daidzein (3), and genistein (4). Further research was conducted on the functions of CHI and HID enzymes, such as... Figure 3 As shown in Figure B, the lack of expression of GmCHI1B1 led to a sharp decrease in the yield of daidzein (3), while GmHID did not significantly affect the synthesis of daidzein (3) and genistein (4). The results of the study on the roles of the three enzymes PAL, C4H, and 4CL in the common pathway of phenylpropane synthesis are shown in [Figure B]. Figure 3 C, compared to other enzyme combinations, only GmPAL2.1, GmCHS7 and GmCHR5 express the most accumulated glycyrrhizin (2) and naringenin (1), indicating that PAL is the rate-limiting enzyme in the common pathway of phenylpropane synthesis and significantly affects the biosynthesis of isoflavones.

[0109] In summary, the results identified the rate-limiting enzymes for the synthesis of various target intermediate compounds of isoflavones in Tobacco Benzoin: the rate-limiting enzymes for the synthesis of naringenin (1) are PAL and CHS, the rate-limiting enzymes for the synthesis of glycyrrhizin (2) are PAL, CHS and CHR, the rate-limiting enzymes for the synthesis of daidzein (3) are PAL, CHS, CHR, CHI and IFS, and the rate-limiting enzymes for the synthesis of genistein (4) are PAL, CHS and IFS.

[0110] Example 2: Screening of highly efficient isoenzymes of rate-limiting enzymes

[0111] Based on the five key rate-limiting enzymes (PAL, CHS, CHR, CHI, and IFS) identified in Example 1 for the upstream isoflavone synthesis pathway, this example cloned isoenzyme genes from different plant sources (mainly leguminous plants) and performed functional verification in *Nicotiana benthamiana* to further increase the yield of the target compounds. The candidate isoenzyme encoding genes are as follows:

[0112] (1) The candidate isozyme encoding genes of PAL include: AtPAL1, AtPAL2 and AtPAL3 from Arabidopsis thaliana; GmPAL1.1, GmPAL2.1 and GmPAL2.3 from soybean; MtPAL from Medicago truncatula; MsPAL from Medicago sativa; TpPAL from Trifolium pratense; TrPAL from Trifolium repens; and NbPAL from Nicotiana benthamiana.

[0113] (2) The candidate isozyme encoding genes of CHS include: GmCHS7 and GmCHS8 from soybean; MtCHS from Medicago truncatula; MsCHS from Medicago sativa; TpCHS from Trifolium pratense; TrCHS from Trifolium repens; and GuCHS from Glycyrrhiza uralensis.

[0114] (3) Candidate isozyme encoding genes for CHR include: GmCHR5, GmCHR1, and GmCHR6 from soybean; MtCHR from Medicago truncatula; MsCHR from Medicago sativa; TpCHR from Trifolium pratense; TrCHR from Trifolium repens; and GuCHR from Glycyrrhiza uralensis.

[0115] (4) Candidate isozyme encoding genes for CHI include: GmCHI1B1, GmCHI1A, and GmCHI2 from soybean; MtCHI from Medicago truncatula; TpCHI from Trifolium pratense; TrCHI from Trifolium repens; and NbCHI from Nicotiana abenthamiana.

[0116] (5) Candidate isozyme encoding genes for IFS include: GmIFS1 from soybean; MtIFS from Medicago truncatula; and GuIFS from Glycyrrhiza uralensis.

[0117] All relevant primer information is shown in Table 1 (serial numbers), and relevant gene information is shown in Table 2. The gene cloning, vector construction, Agrobacterium transformation, and Agrobacterium infection of tobacco leaves involved in Example 2 are the same as in Example 1.

[0118] Using the yield of naringenin (1) as the readout, the catalytic efficiencies of different PAL and CHS were compared; using the yield of glycyrrhizin (2) as the readout, the catalytic efficiencies of different CHR were compared; using the yield of daidzein (3) as the readout, the catalytic efficiencies of different CHI and IFS were compared. While keeping the rate-limiting enzymes involved in the synthesis of other compounds constant, the yields of the target compounds were compared by changing a single enzyme. The results are as follows: Figure 4 As shown. Yield comparisons revealed that AtPAL2 exhibited better catalytic performance among different PALs; GmCHS8 showed good catalytic performance for the synthesis of naringenin (1) and glycyrrhizin (2); the combination of GmCHR5 and GmCHS8 yielded the highest yield of glycyrrhizin (2); GmCHI1B1 yielded the highest yield of daidzein (3); and for different IFSs, GmIFS1 had higher catalytic efficiency than the other two IFSs. The screening of key enzymes and their highly efficient isoenzymes increased the yield of daidzein in *Tobacco Benzoinus* from 0.55 g / kg dry weight to 1.16 g / kg dry weight, and the yield of genistein from 1.08 g / kg dry weight to 1.91 g / kg dry weight.

[0119] Example 3: Screening of multi-gene combinations linked by 2A peptide

[0120] Based on the rate-limiting enzyme identified in Example 1 and the highly efficient isoenzyme screened in Example 2, in order to further increase the yield of daidzein (3) and genistein (4) and simplify some experimental operations, Example 3 utilized a 2A peptide with "self-cleavage" capability as a gene linker to connect multiple target genes, constructing four multiple gene expression vectors (MGVs). The gene combinations included AtPAL2 and GmCHS8 (MGV1), GmCHS8 and GmCHR5 (MGV2), AtPAL2, GmCHR5, and GmCHS8 (MGV3), and GmCHS8, GmCHR5, and GmCHI1B1 (MGV4). A detailed connection diagram is shown below. Figure 5 As shown in Figure A.

[0121] The 2A peptide includes two types: P2A peptide and T2A peptide. The amino acid sequence of P2A peptide is shown in SEQ ID NO.7, and the corresponding nucleotide sequence is shown in SEQ ID NO.8. The amino acid sequence of T2A peptide is shown in SEQ ID NO.9, and the corresponding nucleotide sequence is shown in SEQ ID NO.10.

[0122] The specific process for constructing a multi-gene co-expression vector (single expression cassette) is as follows:

[0123] (1) Construction method of MGV1: 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-AtPAL2 as templates. Using pEAQ-GmCHS8 as a template, the first target gene sequence GmCHS8-P2A-infusion was amplified using primers 37 and 79 and 2×Phanta Max Master Mix of Novizan. The amplification reaction system was the same as in Example 1. Using pEAQ-AtPAL2 as a template, the second target gene sequence was amplified in two steps using primers 20 and 80-81 and 2×Phanta Max Master Mix of Novizan. The reaction system was as follows:

[0124] Step 1: Mix 0.2 μL of P2A-AtPAL2-F primer, 0.2 μL of pEAQ-AtPAL2-R primer, 5 μL of 2×PhantaMax Master Mix, 0.5 μL of plasmid DNA, and 4.1 μL of ddH2O. Then, follow the 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 10 amplification cycles; finally, extend at 72℃ for 5 min, and then use it as a template for the next reaction.

[0125] Step 2: Mix 10 μL of the reaction solution from the previous step, 1 μL of P2A-F primer, 1 μL of pEAQ-AtPAL2-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.

[0126] 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.

[0127] 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 plasmid MGV1: pEAQ-GmCHS8-P2A-AtPAL2.

[0128] (2) Construction method of MGV2: Same as MGV1 method, using pEAQ-GmCHS8 and pEAQ-GmCHR5 as templates, the primers for the first segment of the target gene amplification are numbered 37 and 79, and the primers for the second segment of the target gene amplification are numbered 10, 80 and 82. The final plasmid MGV2 with correct sequencing is obtained: pEAQ-GmCHS8-P2A-GmCHR5.

[0129] (3) Construction method of MGV3: 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 multi-gene expression vectors MGV2 and pEAQ-AtPAL2 constructed above as templates. Using MGV2 as a template, the first target gene sequence GmCHS8-P2A-GmCHR5-T2A-infusion was amplified using primers 37 and 83 and 2×Phanta Max Master Mix of Novizan. The amplification reaction system was the same as in Example 1. Using pEAQ-AtPAL2 as a template, the second target gene sequence was amplified in two steps using the overlap-PCR method with primers 20 and 84-85 and 2×Phanta Max Master Mix of Novizan. The reaction system is as follows:

[0130] Step 1: Mix 0.2 μL of T2A-AtPAL2-F primer, 0.2 μL of pEAQ-AtPAL2-R primer, 5 μL of 2×PhantaMax Master Mix, 0.5 μL of plasmid DNA, and 4.1 μL of ddH2O. Then, follow the 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 10 amplification cycles; finally, extend at 72℃ for 5 min, and then use it as a template for the next reaction.

[0131] Step 2: Mix 10 μL of the reaction solution from the previous step, 1 μL of T2A-F primer, 1 μL of pEAQ-AtPAL2-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.

[0132] 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.

[0133] 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 plasmid MGV3: pEAQ-GmCHS8-P2A-GmCHR5-T2A-AtPAL2.

[0134] (4) Construction method of MGV4: Same as MGV3 method, using MGV2 and pEAQ-GmCHI1B1 as templates. The primers for the first segment of the target gene amplification are serial numbers 37 and 83, and the primers for the second segment of the target gene amplification are serial numbers 12, 84, and 86. The final plasmid MGV4 with correct sequencing is obtained: pEAQ-GmCHS8-P2A-GmCHR5-T2A-GmCHI1B1.

[0135] Functional validation of the above four multi-gene expression vectors was performed by synthesizing daidzein (3) in Nicotiana benthamiana. The results are as follows: Figure 5 As shown in B. The results showed that only the combination of GmCHS8 and GmCHR5 significantly increased the accumulation of daidzein (3) (p-value < 0.05), and also reduced the accumulation of the byproduct genistein (4), while other combinations did not significantly increase the yield of daidzein. In summary, MGV2, namely the combination of GmCHS8-P2A-GmCHR5, can significantly increase the accumulation of daidzein (3), a key intermediate in the isoflavone pathway. This step increased the yield of daidzein in tobacco Benedict from 1.16 g / kg dry weight to 1.78 g / kg dry weight.

[0136] Example 4: Screening of transcription factors to enhance isoflavone biosynthetic flux

[0137] Since flavonoids and isoflavones share the common pathway of phenylpropane synthesis and the precursor naringenin (1), this embodiment introduces transcription factors that can positively regulate upstream biosynthetic genes of flavonoids or inhibitors of isoflavone biosynthetic competitive metabolic pathways (such as anthocyanin biosynthesis) into Tobacco Benzoinus, thereby adjusting the background flavonoid biosynthetic metabolic flux of tobacco to direct more isoflavones.

[0138] First, positive regulators of phenylpropanoid synthesis were screened. Based on previous studies, it was found that the transcription factor combination AmROS1 / AmDEL from snapdragon (Antirrhinum majus), the transcription factor AtMYB12 from Arabidopsis thaliana, and the transcription factors GmMYB12B2 and GmMYB176 from soybean could upregulate some key genes in phenylpropanoid synthesis in plants. These transcription factors (combinations) were then co-expressed with the key gene combination AtPAL2+MGV2+GmCHI1B1+GmIFS1+GmHID(GC8) for daidzein synthesis in Nicotiana benthamiana. The results are as follows: Figure 6 As shown in A, AtMYB12 and GmMYB12B2 both significantly upregulated the accumulation of daidzein (3) and genistein (4).

[0139] Then, anthocyanin inhibitors were screened. Based on previously reported studies, transcription factors AtMYB60, AtMYBL2, and AtCPC in Arabidopsis thaliana and FaMYB1 in strawberry (Fragaria × Ananassa) were found to inhibit a competitive pathway in the isoflavone synthesis pathway, namely the anthocyanin synthesis pathway. These transcription factors were then co-expressed with the key gene combination AtPAL2+MGV2+GmCHI1B1+GmIFS1+GmHID(GC8) for daidzein synthesis in Nicotiana benthamiana. The results showed... Figure 6 B shows that only AtMYB60 resulted in a significant increase in the production of daidzein (3) and genistein (4).

[0140] Further comparisons were made of the effects of single or combined AtMYB12, GmMYB12B2, and AtMYB60 transcription factors on the enhancement of daidzein (3) and genistein (4) yields. Results are as follows... Figure 6As shown in C and 6D, the yields of daidzein (3) under the action of AtMYB12 and AtMYB60 alone were similar and higher than those under the combination of multiple transcription factors. However, AtMYB12 led to the accumulation of more byproduct genistein (4). Therefore, AtMYB60 alone had a better effect on increasing the yield of daidzein (3). For the synthesis of genistein (4), the combination of GmMYB12B2 and AtMYB60 achieved the highest yield. Thus, we finally determined that the optimal gene combination for the synthesis of daidzein (3) is AtPAL2, GmCHS8-P2A-GmCHR5, GmCHI1B1, GmIFS1, GmHID and AtMYB60, and the optimal gene combination for the synthesis of genistein (4) is AtPAL2, GmCHS8, GmIFS1, GmMYB12B2 and AtMYB60. The gene cloning, vector construction, Agrobacterium transformation, and Agrobacterium infection of tobacco leaves involved in this embodiment are the same as in Example 1. The gene information and primers involved are shown in Tables 2 and 1, respectively. This step increased the yield of daidzein in tobacco from 1.78 g / kg dry weight to 4.28 g / kg dry weight, and the yield of genistein from 1.91 g / kg dry weight to 7.17 g / kg dry weight.

[0141] Example 5: Assembly of a gene for efficient isoflavone synthesis

[0142] Based on the optimal gene combination for synthesizing daidzein (3) and genistein (4) determined in Example 4, and to further simplify the operation and lay the foundation for the subsequent synthesis of complex compounds, this example constructs ultra-large plasmids containing three and two gene expression cassettes respectively to assemble the optimal gene combination for synthesizing daidzein (3) and genistein (4). The assembly diagram is shown below. Figure 7 As shown in A, the efficient synthesis of daidzein (3) and genistein (4) was achieved by co-expressing 7 and 5 genes respectively using a single vector.

[0143] Specifically, the process of constructing a multi-gene expression vector (multiple expression cassettes) is as follows:

[0144] 1. First, construct multi-gene expression vectors (single expression cassettes) containing a single expression cassette expressing 2-3 genes, including pEAQ-GmIFS1-P2A-AtMYB60 (MGV5), pEAQ-GmCHIB1-P2A-AtPAL2-T2A-GmHID (MGV7) and pEAQ-GmCHS8-P2A-GmMYB12B2-T2A-AtPAL2 (MGV9).

[0145] 1) Construction method of MGV5: Same as the construction method of MGV1 in Example 3. The templates are pEAQ-GmIFS1 and pEAQ-AtMYB60. The primers for the first segment of the target gene amplification are serial numbers 13 and 105, and the primers for the second segment of the target gene amplification are serial numbers 80, 98 and 106.

[0146] 2) Construction method of MGV7: 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.

[0147] Using pEAQ-GmCHIB1 as a template, the first target gene sequence GmCHIB1-P2A-infusion was amplified using primers 11 and 109 and 2×Phanta MaxMaster Mix of Novizan. The amplification reaction system was the same as in Example 1.

[0148] Using pEAQ-AtPAL2 as a template, primers 80-81 and 110, and 2×Phanta MaxMaster Mix of Novizan, the second target gene sequence was amplified in two steps using overlap-PCR. The reaction system is as follows:

[0149] 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.

[0150] 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.

[0151] Using pEAQ-GmHID as a template, primers 16, 84, and 111, along with 2×Phanta MaxMaster Mix of Novizan, were used to amplify the third target gene sequence in two steps using overlap-PCR. The reaction system is as follows:

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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 plasmid MGV7.

[0156] 3) Construction method of MGV9: Same as MGV7, using pEAQ-GmCHS8, pEAQ-GmMYB12B2, and pEAQ-AtPAL2 as templates. Primers for the first segment of the target gene amplification are numbers 37 and 79, primers for the second segment are numbers 80 and 107-108, and primers for the third segment are numbers 20 and 84-85.

[0157] 2. Based on the above-mentioned multi-gene expression vectors with a single expression cassette, construct multi-gene expression vectors (two expression cassettes) containing two gene expression cassettes expressing 4-5 genes, including pEAQ-35S promoter-GmCHS8-P2A-GmCHR5-NOSteminator-35S promoter-GmIFS1-P2A-AtMYB60-NOS teminator (MGV6, abbreviated as: pEAQ-P35S-GmCHS8-P2A-GmCHR5-TNOS-P35S-GmIFS1-P2A-AtMYB60-TNOS) and pEAQ-35S promoter-GmCHS8-P2A-GmMYB12B2-T2A-AtPAL2-NOS teminator-35Sproutter-GmIFS1-P2A-AtMYB60-NOS teminator (MGV10, abbreviation: pEAQ-P35S-GmCHS8-P2A-GmMYB12B2-T2A-AtPAL2-TNOS-P35S-GmIFS1-P2A-AtMYB60-TNOS).

[0158] 1) Construction method of MGV6: The multi-gene expression vector (single expression cassette) MGV2 was digested with NEB's Asc I restriction endonuclease; using the multi-gene expression vector (single expression cassette) MGV5 as a template, the full-length expression cassette of the vector from the 35S promoter to the NOS terminator was amplified by PCR using primers 112-113 and Novizan's 2×Phanta Max Master Mix, and the amplification reaction system was the same as in Example 1; after recovering the above DNA fragments, single fragments were ligated using Novizan's ClonExpress Ultra One Step Cloning Kit V2 through homologous recombination. The ligation, transformation and plasmid identification were the same as in Example 1, and the plasmid MGV6 was finally obtained.

[0159] Construction method of MGV10: Same as MGV6 method, single enzyme digestion of multi-gene expression vector (single expression cassette) is MGV9, and finally plasmid MGV10 is obtained.

[0160] 3. Based on the above single and two-expression-cassette multi-gene expression vectors, a multi-gene expression vector (three expression cassettes) MGV8 containing three gene expression cassettes expressing seven genes was constructed: pEAQ-35S promoter-GmCHS8-P2A-GmCHR5-NOS teminator-35S promoter-GmIFS1-P2A-AtMYB60-NOS teminator-35Sprout-GmCHIB1-P2A-AtPAL2-T2A-GmHID-NOS terminator (abbreviated as: pEAQ-P35S-GmCHS8-P2A-GmCHR5-TNOS-P35S-GmIFS1-P2A-AtMYB60-TNOS-P35S-GmCHIB1-P2A-AtPAL2-T2A-GmHID-TNOS).

[0161] Construction of MGV8: The multi-gene expression vector (two expression cassettes) MGV6 was digested with NEB's Pac I restriction endonuclease; using the multi-gene expression vector (single expression cassette) MGV7 as a template, the full-length expression cassette from the 35S promoter to the NOS terminator was amplified by PCR using primers 114-115 and Novizan's 2×Phanta Max Master Mix, with the amplification reaction system the same as in Example 1; after recovering the above DNA fragments, single fragments were ligated using Novizan's ClonExpress Ultra One Step Cloning Kit V2 via homologous recombination, with ligation, transformation, and plasmid identification the same as in Example 1, finally yielding plasmid MGV8.

[0162] Production comparison Figure 7 As shown in B and 7C, the yield of daidzein (3) synthesized by a single carrier was slightly lower than that synthesized by multiple carriers, but the difference was not significant, while the yield of genistein (4) was significantly increased. This step increased the genistein yield in *Nicotiana benthamiana* from 7.17 g / kg dry weight to 10.26 g / kg dry weight. Ultimately, the yield of daidzein in *Nicotiana benthamiana* was 4.28 g / kg dry weight, and the yield of genistein was 10.26 g / kg dry weight. Compared with before optimization, the yield of daidzein was increased by approximately 6.8 times, and the yield of genistein was increased by approximately 8.5 times.

[0163] Example 6: Heterogeneous synthesis of various active isoflavone compounds

[0164] Starting from the highly accumulated key isoflavone biosynthetic intermediates daidzein (3) and genistein (4), a wide variety of isoflavone compounds can be synthesized, as shown in the schematic diagram below. Figure 8 As shown. Figure 8 The standard curves for the quantitative determination of daidzein (5), genistein (6), puerarin (7), styracin (8), chickpea extract A (9), and stigmosiderin (10) are as follows: Figure 9 As shown. The gene cloning, vector construction, Agrobacterium transformation, and Agrobacterium infection of tobacco leaves involved in this embodiment are the same as in Example 1. The gene information and primers involved are shown in Table 2 and Table 1, respectively.

[0165] 1. In this embodiment, the genes of soybean glycosyltransferase GmUGT4 and puerarin glycosyltransferase PlUGT43 were further cloned. GmUGT4 was co-expressed in *Nicotiana benthamiana* with the multi-gene expression vectors MGV8 and MGV10 for the synthesis of daidzein and genistein, respectively, to synthesize daidzein (5) and genistein (6). PlUGT43 was co-expressed in *Nicotiana benthamiana* with the multi-gene expression vector MGV10 for the synthesis of puerarin (7). The results are as follows: Figure 10As shown.

[0166] The results showed that daidzein (5) and genistein (6) were synthesized before and after the addition of GmUGT4, and the yields were comparable. It is speculated that the endogenous glycosyltransferases in tobacco Benzoinus are sufficient to meet the needs of daidzein (5) and genistein (6) synthesis, but PlUGT43 can significantly increase the yield of puerarin (7). Finally, the yields of daidzein in tobacco Benzoinus were 18.74 g / kg dry weight, genistein in tobacco was 5.36 g / kg dry weight, and puerarin in tobacco was 4.18 g / kg dry weight.

[0167] 2. In this embodiment, the 2'-hydroxyisoflavone-4'-O-methyltransferase MtHI4'OMT gene from alfalfa was further cloned and co-expressed with the multi-gene expression vectors MGV8 and MGV10 for the synthesis of daidzein and genistein in tobacco to synthesize methylated isoflavones argentin (8) and chickpea ginsenoside A (9).

[0168] Meanwhile, this embodiment constructed a GmHID-free multi-gene expression vector MGV12 (pEAQ-35S promoterGmCHS8-P2A-GmCHR5-NOS teminator-35S promoter-GmIFS1-P2A-AtMYB60-NOS teminator-35S promoter-GmCHIB1-P2A-AtPAL2-NOS terminator). The MGV12 construction method is as follows: First, construct the multi-gene expression vector (single expression cassette) pEAQ-GmCHIB1-P2A-AtPAL2 (MGV11): the method is the same as the MGV1 construction method in Example 3, the templates are pEAQ-GmCHIB1 and pEAQ-AtPAL2, the first target gene amplification primers are serial numbers 11 and 109, and the second target gene amplification primers are serial numbers 20 and 80-81. Then, the multi-gene expression vector (two expression cassettes) MGV6 was digested with NEB's Pac I restriction endonuclease. Using the multi-gene expression vector (single expression cassette) MGV11 as a template, the full-length expression cassette from the 35S promoter to the NOS terminator was amplified by PCR using primers 114-115 and Novizan's 2×Phanta Max Master Mix. The amplification reaction system was the same as in Example 1. After recovering the above DNA fragments, single fragments were ligated using Novizan's ClonExpress Ultra One Step Cloning Kit V2 via homologous recombination. The ligation, transformation, and plasmid identification were the same as in Example 1, and the plasmid MGV12 was finally obtained. Subsequently, the 2'-hydroxyisoflavone-4'-O-methyltransferase MtHI4'OMT gene from Alfalfa was cloned and co-expressed with the multi-gene expression vector MGV12 in Nicotiana benthamiana to synthesize the methylated isoflavones gentiopicrin (8) and chickpea ginseng A (9).

[0169] The results are as follows Figure 11 As shown, it was found that co-expression of daidzein (3) and genistein (4) with MtHI4'OMT using the GmHID-free multi-gene expression vectors MGV12 and MGV10 could convert them into genistein (8) and chickpea extract A (9), respectively, and obtain a certain yield. However, co-expression of MtHI4'OMT using the GmHID-containing MGV8 significantly reduced the yield of genistein (8) and chickpea extract A (9). Finally, the yield of genistein in *Nicotiana benthamiana* was 2.26 g / kg dry weight, and the yield of chickpea extract A was 4.08 g / kg dry weight.

[0170] 3. In this embodiment, the genes encoding isoflavone 2'-hydroxylase MtI2'H, isoflavone reductase MsIFR, vesitone reductase MsVR, and santalin synthase MtPTS were further cloned and expressed in tobacco leaves in a pyramidal combination sequence: MGV8, MGV8+MtHI4'OMT, MGV8+MtHI4'OMT+MtI2'H, and MGV8+MtHI4'OMT+MtI2'H. The biosynthetic pathway of santalin (10) was reconstructed in *Nicotiana benthamiana* using the sequences H+MsIFR, MGV8+MtHI4'OMT+MtI2'H+MsIFR+MsVR, MGV8+MtHI4'OMT+MtI2'H+MsIFR+MsVR+MtPTS, and MGV12+MtHI4'OMT+MtI2'H+MsIFR+MsVR+MtPTS, and santalin (10) was successfully synthesized. Figure 12 As shown, when MGV8+MtHI4'OMT+MtI2'H+MsIFR+MsVR+MtPT is expressed in Tobacco Benedictine, the amount of santalin (10) is the highest, and the final yield of santalin in Tobacco Benedictine is 0.72 g / kg dry weight.

[0171] This invention provides a method for constructing and applying a Benzodiacetic tobacco chassis for synthesizing isoflavone compounds. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. 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 method for constructing a Benzodiacetic tobacco chassis for synthesizing isoflavone compounds, characterized in that, The first Nicotiana benthamiana chassis for synthesizing isoflavone compounds was constructed by co-expressing the encoding genes of phenylalanine ammonia-lyase PAL, chalcone synthase CHS, chalcone reductase CHR, chalcone isomerase CHI, isoflavone synthase IFS, 2-hydroxyisoflavone dehydratase HID and transcription factors into Agrobacterium-mediated Nicotiana benthamiana using an expression vector. Alternatively, by using an expression vector, the encoding genes of phenylalanine ammonia-lyase PAL, chalcone synthase CHS, isoflavone synthase IFS, and transcription factors can be co-expressed into Agrobacterium-mediated Nicotiana benthamiana to construct a second Nicotiana benthamiana chassis for synthesizing isoflavone compounds. The coding genes are linked by a 2A peptide.

2. The construction method according to claim 1, characterized in that, The encoding genes for phenylalanine ammonia-lyase PAL include any one of AtPAL1, AtPAL2, AtPAL3, GmPAL1.1, GmPAL2.1, GmPAL2.3, MtPAL, MsPAL, TpPAL, TrPAL, and NbPAL; the encoding genes for chalcone synthase CHS include any one of GmCHS7, GmCHS8, MtCHS, MsCHS, TpCHS, TrCHS, and GuCHS; the encoding genes for chalcone reductase CHR include GmCHR5, GmCHR1, GmCHR6, MtC The chalcone isomerase CHI is any one of HR, MsCHR, TpCHR, TrCHR, and GuCHR; the gene encoding the chalcone isomerase CHI includes any one of GmCHI1B1, GmCHI1A, GmCHI2, MtCHI, TpCHI, TrCHI, and NbCHI; the gene encoding the isoflavone synthase IFS includes GmIFS1, MtIFS, and GuIFS; the gene encoding the 2-hydroxyisoflavone dehydratase HID is GmHID; the transcription factor includes any one or a combination of AtMYB12, GmMYB12B2, and AtMYB60.

3. The construction method according to claim 2, characterized in that, The gene encoding the phenylalanine ammonia-lyase PAL is AtPAL2; the gene encoding the chalcone synthase CHS is GmCHS8; the gene encoding the chalcone reductase CHR is GmCHR5; the gene encoding the chalcone isomerase CHI is GmCHI1B1; the gene encoding the isoflavone synthase IFS is GmIFS1; and the transcription factors are GmMYB12B2 and / or AtMYB60.

4. The construction method according to claim 3, characterized in that, When the constructed *Nicotiana benthamiana* chassis is the first *Nicotiana benthamiana* chassis, the transcription factor is AtMYB60; Alternatively, when the constructed *Tobacco Bungei* chassis is a second *Tobacco Bungei* chassis, the transcription factors are GmMYB12B2 and AtMYB60.

5. The construction method according to claim 1, characterized in that, The 2A peptide is any one or a combination of two of the P2A peptide or the T2A peptide.

6. The construction method according to claim 4, characterized in that, The amino acid sequence of the P2A peptide is shown in SEQ ID NO.7, and the corresponding nucleotide sequence is shown in SEQ ID NO.8; the amino acid sequence of the T2A peptide is shown in SEQ ID NO.9, and the corresponding nucleotide sequence is shown in SEQ ID NO.

10.

7. The construction method according to claim 1, characterized in that, The expression vector is pEAQ-HT; the co-expression into Agrobacterium-mediated Tobacco Benzoenta specifically involves co-expressing the expression vector containing the coding gene into Agrobacterium, and then using Agrobacterium to infect Tobacco Benzoenta leaves.

8. The construction method according to claim 7, characterized in that, The expression vector containing the coding gene is a single-gene expression vector or a multi-gene expression vector containing multiple expression cassettes.

9. The construction method according to claim 8, characterized in that, When the constructed *Nicotiana benthamiana* chassis is the first *Nicotiana benthamiana* chassis, the single-gene expression vectors are pEAQ-AtPAL2, pEAQ-GmCHS8, pEAQ-GmCHR5, pEAQ-GmCHI1B1, pEAQ-GmIFS1, pEAQ-GmHID, and pEAQ-AtMYB60; the multi-gene expression vector containing multiple expression cassettes is the multi-gene expression vector MGV8 containing three expression cassettes: pEAQ-35S promoter-GmCHS8-P2A-GmCHR5-NOS teminator-35S promoter-GmIFS1-P2A-AtMYB60-NOS teminator-35S promoter-GmCHIB1-P2A-AtPAL2-T2A-GmHID-NOS terminator; Alternatively, when the constructed *Nicotiana benthamiana* chassis is a second *Nicotiana benthamiana* chassis, the single-gene expression vector is pEAQ-AtPAL2, pEAQ-GmCHS8, pEAQ-GmIFS1, pEAQ-GmMYB12B2, and pEAQ-AtMYB60; the multi-gene expression vector containing multiple expression cassettes is the multi-gene expression vector MGV10 containing two expression cassettes: pEAQ-35S promoter-GmCHS8-P2A-GmMYB12B2-T2A-AtPAL2-NOS teminator-35S promoter-GmIFS1-P2A-AtMYB60-NOS teminator.

10. The application of the *Tobacco Benzoinus* chassis constructed by the method according to any one of claims 1 to 9 in the synthesis of isoflavone compounds.

11. The application according to claim 10, characterized in that, The isoflavone compounds include any one or a combination of several of the following: daidzein, genistein, daidzein, genistein, puerarin, methylated isoflavone gentiopicrin, chickpea ginsenoside A, and mediopterygium oleracea.

12. The application according to claim 11, characterized in that, (i) Synthesizing daidzein using the first Benedictine chassis; or, (ii) Synthesize lignin dyes using the second Benedict's tobacco chassis; or, (iii) Using the first Nicotiana benthamian carcass as the starting carcass, daidzein is synthesized by overexpressing the glycosyltransferase GmUGT4; or... (iiii) Using the second Nicotiana benthamiana chassis as the starting chassis, overexpressing the glycosyltransferase GmUGT4 to synthesize genistein; or... (iv) Using the first Nicotiana benthamian carcass as the starting carcass, puerarin was synthesized by overexpressing the glycosyltransferase PlUGT43. or, (v) Using the first Benedictine chassis as the starting chassis, methylated isoflavone argentin was synthesized by overexpressing 2'-hydroxyisoflavone-4'-O-methyltransferase MtHI4'OMT. or, (vi) Using the second Benedictine chassis as the starting chassis, 2'-hydroxyisoflavone-4'-O-methyltransferase MtHI4'OMT was overexpressed to synthesize chickpea protein A; or, (vii) Using the first Benedictine chassis as the starting chassis, 2'-hydroxyisoflavone-4'-O-methyltransferase MtHI4'OMT, isoflavone 2'-hydroxylase MtI2'H, isoflavone reductase MsIFR, vestitone reductase MsVR and santalin synthase MtPTS were overexpressed to synthesize santalin.

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