CN114891807B

CN114891807B - Application of CYP450 gene in regulating and controlling soybean isoflavone synthesis

Info

Publication number: CN114891807B
Application number: CN202210596380.4A
Authority: CN
Inventors: 庞永珍; 夏亚迎
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2022-05-30
Filing date: 2022-05-30
Publication date: 2024-01-26
Anticipated expiration: 2042-05-30
Also published as: CN114891807A

Abstract

The invention provides an application of CYP450 gene in regulating soybean isoflavone synthesis, wherein the gene is at least one of the following genes: CYP93A1, CYP93A2 and CYP82D26 genes. The CYP93A1, CYP93A2 and CYP82D26 genes are endoplasmic reticulum localization. The amino acid sequence of the CYP450 gene comprises an E-R-R triplet (E-R-R triplet) domain and a heme binding domain, wherein the E-R-R triplet is composed of a K-helix conserved sequence (KETLR) and an LR column conserved sequence. The invention provides application of CYP93A1, CYP93A2 and CYP82D26 genes in regulating and controlling soybean isoflavone synthesis for the first time, and discloses a molecular mechanism of CYP93A1, CYP93A2 and CYP82D26 enzyme catalysis. These results enrich the diversity of CYP450 enzyme catalytic mechanisms and reflect the diversity and complexity of soy isoflavone biosynthetic pathways.

Description

Application of CYP450 gene in regulating and controlling soybean isoflavone synthesis

Technical Field

The invention relates to application of CYP450 genes, in particular to application of CYP450 genes in regulating and controlling soybean isoflavone synthesis, and belongs to the technical field of genetic engineering application.

Background

The soybean is annual dicotyledonous herb plant of the genus Glycine of the subfamily Papilionaceae. The soybean has rich nutrition, and is rich in proteins, lipid substances, carbohydrates, vitamins, minerals, and various bioactive substances. The soybeans have the nutritive values of promoting intestinal health, reducing blood sugar and blood fat, preventing cancers, strengthening brain, improving intelligence and the like, and are often processed into bean products of different types for eating by people. In addition, soybeans have a large variety of bioactive substances, and mainly contain isoflavones, saponins, sterols, phospholipids, and the like. Isoflavones have various pharmacological properties such as antioxidant, antiinflammatory, antiviral, anticancer, and prophylactic effects on climacteric, osteoporosis, cardiovascular and cerebrovascular diseases, and diabetes. Isoflavone is also a low molecular weight antibacterial compound, has broad-spectrum antibacterial effect on pathogenic bacteria, and can be used as a signal molecule to play an important role in interaction of plants and microorganism molecules.

The main components of soybean isoflavone are Daidzein, genistein, glycitein, and three kinds of glycosides, malonyl glycoside and acetyl glycoside. However, the biosynthetic pathway of soybean isoflavone is not perfect, wherein the enzyme in the biosynthetic pathway of soybean isoflavone end belongs to CYP450 enzyme, but the encoding gene is not clear, and whether other CYP450 genes are involved in the biosynthetic pathway of soybean isoflavone needs to be further excavated.

Disclosure of Invention

The invention aims to provide application of CYP93A1, CYP93A2 and CYP82D26 genes in regulating and controlling soybean isoflavone synthesis, and discloses a molecular mechanism of CYP93A1, CYP93A2 and CYP82D26 enzyme catalysis. These results enrich the diversity of the CYP450 enzyme catalytic mechanisms, as well as the diversity and complexity of the soy isoflavone biosynthetic pathways.

According to one aspect of the present application there is provided the use of a CYP450 gene for modulating soy isoflavone synthesis, the gene being selected from at least one of the following genes: CYP93A1, CYP93A2 and CYP82D26 genes.

Specifically, the CYP93A1, CYP93A2, and CYP82D26 genes are endoplasmic reticulum localization.

Specifically, the amino acid sequence of the CYP450 gene comprises an E-R-R triplet (E-R-R triade) domain and a heme binding domain, wherein the E-R-R triplet is composed of a K-helix conserved sequence (KETLR) and an LR column conserved sequence.

Specifically, the CYP93A1 and CYP93A2 gene coded proteins have catalytic activity on 3, 9-dihydroxypterocarpine, and can catalyze the 3, 9-dihydroxypterocarpine to generate daidzein.

In some embodiments, the protein encoded by the CYP93A2 gene has catalytic activity on glycyrrhizin, and can catalyze the production of daidzein from glycyrrhizin.

In some embodiments, the protein encoded by the CYP82D26 gene has catalytic activity on naringenin, and is capable of catalyzing the production of daidzein from naringenin.

According to another aspect of the present application there is also provided the use of a CYP450 gene for modulating daidzein synthesis, the gene being selected from at least one of the following genes: CYP93A1, CYP93A2 and CYP82D26 genes.

According to another aspect of the present application there is also provided the use of a CYP450 gene in the cultivation of a transgenic plant, the gene being selected from at least one of the following genes: CYP93A1, CYP93A2 and CYP82D26 genes.

In some embodiments, the transgenic plant produces soy isoflavones.

In some embodiments, the transgenic plant is selected from soybean or a close species thereof.

In some embodiments, the CYP93A1 and CYP93A2 genes are located predominantly downstream of the daidzein gene in the soybean body.

In some embodiments, the natural substrate of the protein encoded by the CYP82D26 gene comprises naringenin and a compound downstream of daidzein.

The beneficial effects of the invention are as follows:

1. the invention provides the application of CYP93A1, CYP93A2 and CYP82D26 genes in soybean isoflavone synthesis for the first time, and discloses the molecular mechanism of CYP93A1, CYP93A2 and CYP82D26 enzyme catalysis.

2. The invention discovers that CYP93A1 and CYP93A2 can catalyze 3, 9-dihydroxypterocarpine to generate daidzein which is respectively positioned at the upstream of the metabolic pathway and Glycinol which is respectively positioned at the downstream of the metabolic pathway. CYP82D26 which can be dehydroxylated and has isoflavone synthetase function is also discovered for the first time, and a catalytic product of the CYP82D26 is daidzein.

3. The in vivo results of the present invention demonstrate that the natural substrate for CYP82D26 may not only be naringenin, but may also be catalytically active against substances downstream of daidzein.

4. The in vivo results of the present invention show that the CYP93A1, CYP93A2 and CYP82D26 genes can synthesize soybean isoflavone in plants.

Drawings

FIG. 1 is a phylogenetic tree of CYP81, CYP82 and CYP93 proteins in soybean;

FIG. 2 is a graph showing the expression profile of different family genes in different tissues;

FIG. 3 is a multiple sequence alignment of amino acid sequences of the CYP93 family;

FIG. 4 is an enzyme activity assay for the heterologous expression of CYP93A1, CYP93A2 and CYP93A3 in yeast fed with 3, 9-dihydroxypterocarpine as a substrate; (a) an enzyme activity product using empty vector pYeDP60 as a negative control; (B-D) are the enzymatic products of yeast strains that heterologously express CYP93A1, CYP93A2, and CYP93A3, respectively; (E) a3, 9-dihydroxypterocarpine standard; (F) a daidzein standard;

FIG. 5 is an ultraviolet absorbance spectra analysis of the peaks P1 and P2 of the enzyme activity products of CYP93A1 and CYP93A2 catalyzed 3, 9-dihydroxypterocarpine; (a) uv absorbance profile of product peak P1; (B) Ultraviolet absorption spectrum of 3, 9-dihydroxypterocarpine standard; (C) uv absorbance profile of product peak P2; (D) ultraviolet absorbance profile of daidzein;

FIG. 6 is UPLC/MS and UPLC/MS/MS identification of the enzyme activity products P1 and P2 of CYP93A1 and CYP93A2 catalyzed 3, 9-dihydroxypterocarpine; primary mass spectrum (left), secondary mass spectrum (right). (a) a standard for 3, 9-dihydroxypterocarpine; (B-C) product peaks P1 and P2; (D) a daidzein standard;

FIG. 7 shows the conversion of CYP93A1, CYP93A2 and CYP93A3 to glycerol and daidzein by catalyzing the production of 3, 9-dihydroxypterocarpan;

FIG. 8 is an enzyme activity assay for the heterologous expression of CYP93A1, CYP93A2 and CYP93A3 in yeast fed with glycyrrhizin as a substrate; (a) an enzyme activity product using empty vector pYeDP60 as a negative control; (B-D) are the enzymatic products of yeast strains that heterologously express CYP93A1, CYP93A2, and CYP93A3, respectively; (E) a glycyrrhizin standard; (F) a daidzein standard;

FIG. 9 is a graph showing the result of ultraviolet absorbance analysis of peak P of enzyme activity product of CYP93A1 catalyzed glycyrrhizin; (a) uv absorbance profile of product peak P; (B) ultraviolet absorbance profile of daidzein;

FIG. 10 shows the results of UPLC/MS and UPLC/MS/MS identification of the peak P of enzyme activity product of CYP93A1 catalyzed glycyrrhizin; primary mass spectrum (left), secondary mass spectrum (right): (a) product peak P; (B) a daidzein standard;

FIG. 11 shows the conversion of glycyrrhizin to daidzein by CYP93A1, CYP93A2 and CYP93A 3;

FIG. 12 is a structural model of the docking of 3, 9-dihydroxypterocarpine and HEM with CYP93A1, CYP93A2 and CYP93A3 protein molecules;

FIG. 13 is subcellular localization of CYP93A1 and CYP93A2 in tobacco leaf epidermis; (A) fluorescent signal of CYP93A1-GFP fusion protein. (B) fluorescent signal of CYP93A2-GFP fusion protein. (C) Control pCAMBIA1302-GFP fluorescence signal, scale = 25 μm;

FIG. 14 is a tissue expression profile of CYP93A1, CYP93A2, and CYP93A 3;

FIG. 15 is a process of producing soybean hairy roots;

FIG. 16 is a screen of CYP93A1 and CYP93A2 transgenic hairy roots;

FIG. 17 is an analysis of daidzein and genistein content in CYP93A1 and CYP93A2 transgenic hairy roots;

FIG. 18 is an analysis of daidzein and genistein content in CYP93A1 and CYP93A2 transgenic hairy roots;

FIG. 19 is an enzyme activity assay for the heterologous expression of CYP82D26 in yeast fed with naringenin as a substrate; (a) naringenin standard; (B) an enzyme activity product using empty vector pYeDP60 as a negative control; (C) an enzyme activity product of a yeast strain that heterologously expresses CYP82D 26; (D) a daidzein standard;

FIG. 20 is an ultraviolet absorbance spectrum analysis of peak P of enzyme activity product of CYP82D26 catalyzed naringenin; a) Ultraviolet absorbance spectrum of enzyme activity product peak P; (B) ultraviolet absorption spectrum of daidzein as standard;

FIG. 21 is UPLC/MS and UPLC/MS/MS identification of the enzyme activity product P of CYP82D26 catalyzed naringenin; (A) Primary and secondary mass spectra of the product peak; (A) A primary mass spectrum and a secondary mass spectrum of the standard daidzein;

FIG. 22 is a graph showing the conversion of naringenin to daidzein catalyzed by CYP82D 26;

FIG. 23 is subcellular localization of CYP82D26 in tobacco; (A) fluorescent signal of CYP82D26 fusion protein. (B) Control pCAMBIA1302-GFP fluorescence signal, scale = 25 μm;

FIG. 24 is a tissue expression profile of CYP82D 26;

FIG. 25 is a structural model of naringenin and HEM in molecular docking with CYP82D26 protein;

FIG. 26 is a screen of CYP82D26 transgenic hairy roots; (a) highly expressed hairy roots; (B) low-expressing hairy roots;

FIG. 27 is an analysis of total flavone, daidzein and genistein content in CYP82D26 transgenic hairy roots.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 screening of CYP450 Gene in soybean isoflavone biosynthesis pathway

1. Experimental method

(1) Downloading a soybean CYP450 family classification naming file from a Cytochrome P450 Homepage website (https:// drnelson.uthsc.edu /), and downloading a gene CDS sequence and a nucleotide sequence from a phytozome website (https:// phytozome-next.jgi.doe.gov/pz/portal.htmL);

(2) Downloading soybean gene expression spectrum data from a soybase (https:// soybase. Org/soyseq /) website, normalizing the data by a z-score normalization method, and then drawing a heat map by TBtools software;

(3) Analyzing the GEO data set GDS3242 and GDS3244 on NCBI to count the condition that soybean CYP450 gene is induced to express by phytophthora sojae;

(4) Carrying out advanced structure prediction of the protein at an I-TASSER website (https:// zhanggroup. Org// I-TASSER /) and obtaining a protein three-dimensional structure model of homology modeling;

(5) Downloading small molecular compounds from a Pubchem website (https:// Pubchem. Ncbi. Nlm. Nih. Gov /), and converting the formats of the small molecular compounds by using OpenBabel-2.4.1 software;

(6) Performing molecular Docking (dock) on the homology-modeled protein tertiary structure model and the target compound by using Autodock4.0, and presenting a molecular Docking model diagram by using PyMOL software;

(7) Homologous sequence alignment was performed using ClustalX, and p-distance model, pairwise deletion and 1,000Bootstrap were selected to construct a Neighbor-joining (NJ) evolutionary tree using Mega-X software.

2. Analysis of results

1. The total of 332 CYP450 genes in soybean, 10 Clan, is divided into 48 families. Being classified into type a and non-type a, type a CYPs 450 tend to be involved in the synthesis of secondary metabolites (e.g., phenylpropanes, etc.), whereas non-type a CYPs 450 tend to be involved in the synthesis of primary metabolites (e.g., sterols, fatty acids, etc.), as well as hormones and other signaling molecules. Flavonoids are an important plant secondary metabolite, and in the biosynthetic pathway of flavonoids, there are many enzymes belonging to the CYP450 enzymes, which are mainly the CYP75 family, the CYP93 family, the CYP71D, CYP E and the CYP82D subfamily.

The present study found that CYP450 genes in the same metabolic pathway function similarly and their expression patterns are similar. In addition, after soybean is infected by phytophthora sojae, a large amount of isoflavone compounds are enriched, wherein the expression level of C4H, IFS, I2S energy, P6S energy and Cyclase (S) which are involved in the isoflavone biosynthesis pathway are up-regulated after being induced by the phytophthora sojae. Based on these characteristics, in order to effectively screen CYP450 genes involved in soybean isoflavone biosynthesis pathway, the present study sets the following three screening conditions: first, candidate CYP450 genes belong to the CYP450 family that is related to flavone metabolism or is evolutionarily similar. Second, the candidate CYP450 gene has a similar gene expression pattern to that of its genes reported in this pathway (e.g., IFS, HID, P6D. RTM., G4DT and G2 DT). Third, the expression level of the candidate CYP450 gene needs to be induced by Phytophthora sojae.

The present study reconstructs amino acid sequences of soybean CYP81, CYP82 and CYP93 families by an adjacent-junction (NJ) method into 12 genes of the CYP81 family in the soybean of the evolutionary tree (fig. 1), wherein 11 belongs to the CYP81E subfamily, which may function similarly to the reported genes, encode group I2, or group I3; the CYP82 family has 24 genes in total, the functions of the CYP82 family are unknown, and the CYP82 family accords with the first condition of screening in the study, so the study takes the CYP82 family as a candidate family focused by the study. Furthermore, this study found that there are four subfamilies in the CYP93 family, with the CYP93A subfamily containing 8 genes, and only two genes for each of the CYP93B and CYP93C subfamilies, and only one gene for CYP 93E. Members of the CYP93B and CYP93C subfamilies, as well as the enzymes encoded by CYP93A1, are involved in soy isoflavone synthesis, while the function of most members of the most-formed CYP93A subfamilies is not yet known and meets the first screening criteria of the study, so the study also targets members of the CYP93A subfamilies with unknown function.

Analysis of the expression profiles of the genes by using the gene expression data on the SoyBase website shows that the HID, the IFS2 (CYP 93C1v 2) and the G4DT have the highest expression level in roots and lower expression levels in other tissues; IFS1 (CYP 93C 5) was expressed in the highest amount in roots, and seeds were used for 42 days after flowering; p6 seeds (CYP 93A 1) are expressed in the highest amount in root nodules and secondary in roots; and the expression level of both G2DT was higher in both roots and flowers (FIG. 2). The 7 genes of CYP93A1, CYP93A2, CYP93A3, CYP93A19, CYP93A24, CYP93A26 and CYP93A30 in the CYP93A subfamily are all the highest in the expression level in roots, and the expression level in other tissues is lower, so that the gene is in accordance with the second screening standard of the study; the expression level of CYP93A41 is highest in seeds 14 days after flowering, and the expression level of CYP93E1 in young leaves, pods, seeds 35 days after flowering and seeds 42 days after flowering is higher, which is not in accordance with the second screening standard of the study.

In the CYP82 family, the 4 genes CYP82A3, CYP82A23, CYP82D26 and CYP82D29 are the highest in the root, the expression level of other tissues is lower, the expression level of CYP82A22 and CYP82C20 is the highest in the root, the expression level of other tissues is lower, and the modes of the 6 genes are similar to the expression modes of HID, IFS and G4DT, so that the second screening standard of the study is met. The CYP82A24 has the highest expression level in roots, the second highest expression level in flowers, and lower expression level in other tissues, and the expression pattern of the CYP82A24 is similar to that of G2DT1, and meets the second screening standard of the study. The expression levels of the genes CYP82A2, CYP82A4, CYP82A18, CYP82A19, CYP82A20, CYP82A26, CYP82C18, CYP82C21, CYP82D27, CYP82D28, CYP82L4 and CYP82L6 in flowers are the highest, and the expression patterns are similar to the expression patterns of G2DT2, and meet the second screening standard of the study. The expression level of CYP82A25 in flowers and seeds is higher, the expression level of CYP82C1 and CYP82D25 in flowers, pods and seeds is higher, the expression level of CYP82J3 in flowers and pods is higher, the expression level of CYP82L5 in all tissues is lower, and the 5 genes do not accord with the second screening standard of the study.

By tissue expression profiling, the present study focused on the conditional 7 genes of the CYP93A1, CYP93A2, CYP93A3, CYP93A19, CYP93A24, CYP93A26 and CYP93A30, and 19 CYP82A2, CYP82A3, CYP82A4, CYP82a18, CYP82a19, CYP82a20, CYP82a22, CYP82a23, CYP82a24, CYP82a26, CYP82C18, CYP82C20, CYP82C21, CYP82D26, CYP82D27, CYP82D28, CYP82D29, CYP82L4 and CYP82L 6.

Then, the present study further analyzed these 25 genes using the chip data on NCBI, and found that the expression levels of HID, IFS1 (CYP 93C 5), IFS2 (CYP 93C1v 2), CYP93A1 (P6P 9), G4DT and two G2DT were increased 3-8 fold by using the Phytophthora sojae induction expression (as shown in Table 1), and the third screening condition of the present study was satisfied. In addition, in the CYP93 family, the expression level of CYP93A1 and CYP93A41 is increased by 5 times, CYP93A2 is increased by 3 times and CYP93A3 is increased by 6 times after being treated by phytophthora sojae, which accords with the third screening condition of the study, but the CYP93A41 in the four genes does not accord with the second screening condition of the study; and the expression quantity of CYP82A2 and CYP82A23 in the CYP82 family is increased by 5 times after being treated by phytophthora sojae, the expression quantity of CYP82A3 and CYP82C20 is increased by two times, the expression quantity of CYP82A4 is increased by 7.5 times, and the expression quantity of CYP82D26 is increased by 4.5 times, so that the third screening condition of the research is met. Meanwhile, the present study also analyzed the CYP81 family genes, found that 7 CYP81 family genes are induced by Phytophthora sojae, the expression level is increased by 5-8 times, and they all belong to CYP81E subfamilies, which is consistent with the prediction of the present study, because currently reported CYP81E family genes are all functions of I2-encoding group or I3-encoding group, while CYP93 family genes are various in functions, and CYP82 family genes are less reported in functions, so the present study focuses on CYP93 and CYP82 families.

Finally, the study treated 8 genes of both families, which met these three screening criteria simultaneously, as candidate genes (see table 1), including CYP93A2, CYP93A3, CYP82A2, CYP82A3, CYP82A4, CYP82a23, CYP82C20 and CYP82D 26.

TABLE 1 analysis of expression level of target Gene after Phytophthora sojae

Example 2 functional analysis of CYP93 family and CYP82 family candidate genes

1. Experimental method

1. Extraction method of soybean total RNA

The mortar used in the milling process of this experiment was ignited with absolute ethanol to remove RNase. Centrifuge tubes and tips were used for the experiments, all RNase free.

(1) Taking a proper amount of plant materials, quickly grinding the plant materials into powder after quick freezing by liquid nitrogen, weighing about 100mg of powder, and filling the powder into a 1.5mL RNase free centrifuge tube;

(2) 1mL TRIzol-A was added ⁺ Vortex shaking for 30s, standing at room temperature for 5min;

(3) About 0.2mL of chloroform was added thereto, and the mixture was vortexed and shaken for 30 seconds, left at room temperature for 5 minutes, and then centrifuged at 12,000rpm at 4℃for 10 minutes. The mixed solution is divided into three layers: the upper layer is an aqueous phase containing RNA, the middle layer contains protein, and the lower layer contains material residues and an organic phase of DNA;

(4) Transferring 500 μl of supernatant into a new centrifuge tube of 1.5mL RNase free, adding isopropanol with equal volume, slowly mixing upside down, and standing at room temperature for 30min;

(5) Centrifuging at 12,000rpm and 4deg.C for 10min;

(6) Discarding the supernatant, placing RNA in a tube bottom in a gelatinous precipitate, adding 1mL of 75% ethanol to wash the precipitate, centrifuging at 12,000rpm and 4 ℃ for 10min, and repeating the step (6);

(7) Centrifuging the RNase free centrifuge tube with the supernatant for a short time, sucking out residual ethanol with a pipette, opening a tube cover, and drying the precipitate at room temperature for 5-7min;

(8) Adding 30-50 mu L of H2O treated by DEPC to dissolve RNA, and storing in a refrigerator at-20 ℃ for standby.

2. cDNA synthesized by rapid reverse transcription

cDNA was synthesized according to the Fast King gNDADispelling RT Super Mix protocol.

3. Cloning of full-Length Gene

PCR reactions were performed using soybean 2 week old root cDNA as template and Phusion high fidelity enzyme instructions.

4. Recovery of fragments of interest

The recovery fragments with high purity and high concentration are obtained by using the recovery kit instruction book of kang for century gelatin for operation.

5. Cleavage of vector and fragment of interest

According to the sequence of each gene, a proper enzyme cutting site is selected to design a primer, and the primer is operated by a corresponding restriction enzyme. The enzyme digestion operation is strictly carried out according to the instruction of the selected restriction enzyme.

6. Ligation of the CYP450 fragment of interest with the Yeast expression vector pYeDP60

According to T ₄ In the specification of DNA ligase, a ligation reaction system shown in Table 6 is established between a vector recovered by gel and DNA fragments after enzyme digestion in a sterile centrifuge tube, and the mixture is gently stirred and mixed, and reacted for 8-14h at 16 ℃.

7. Ligation of the CYP450 fragment of interest to the plant expression vector pK7WG2D/pK7 GWIGWG 2 II 2D

The reaction system was added as in Table 2, incubated in a constant temperature heater at 25℃for 1h, the reaction product transformed E.coli DH 5. Alpha. And screened on LB plates containing kan, and the vector plasmid was selected for monoclonal identification, sequenced and stored for further use.

TABLE 2 BP reaction system of Gateway system

The reaction system was added according to Table 3, incubated at 25℃for 2 hours in a constant temperature heater, 0.25. Mu.L of Proteinase K was added, and the reaction was terminated by treatment in a water bath at 37℃for 10 minutes. The reaction product was transformed into E.coli DH 5. Alpha. And screened on a Spec-containing LB plate, and the vector plasmid was selected for monoclonal identification, sequenced and stored for use.

TABLE 3 LR reaction System of Gateway System

8. Ligation of the CYP450 fragment of interest to the pCAMBIA1302 vector

The pCAMBIA1302 vector was double digested with Nco I and SpeI to prepare a digestion system, which was then reacted at 37℃for 3 hours. After the pCAMBIA1302 vector is subjected to enzyme digestion by using Nco I and SpeI, the enzyme digestion product and the CYP450 PCR product are recovered by glue, and are connected by a seamless cloning method. And 5 mu L of the reaction product is transformed into escherichia coli DH5 alpha, and the obtained product is screened on an LB plate containing kan, and the monoclonal identification, sequencing and vector plasmid preservation are carried out for standby.

9. In vitro substrate feeding yeast

(1) Selecting transgenic positive yeast monoclonal and culturing in 100 μl SGI+Trp (containing 2% glucose) liquid culture medium at 28deg.C under shaking until OD ₆₀₀ About 1 to about 2;

(2) Inoculating 1/100 of the strain into 1mL of fresh SGI+Trp (containing 2% glucose) liquid culture medium, and performing shake culture at 28 ℃ for 12h;

(3) 8,000rpm, centrifuging for 10min, discarding the supernatant, and resuspending the bacterial pellet with 1mL fresh SGI+Trp (2% galactose in) liquid medium;

(4) Adding substrate with final concentration of 10 mu M into the re-suspension bacteria liquid, and carrying out shaking culture at 28 ℃ for 24 or 48 hours;

(5) Adding 1mL of ethyl acetate, oscillating for 30s, and performing ultrasonic treatment for 30min;

(6) 8000rpm, centrifuging for 10min, and placing the supernatant into a 2mL centrifuge tube;

(7) Repeating steps 5 and 6, centrifuging the supernatant obtained in the two steps at 12,000rpm for 5min, and collecting the supernatant;

(8) After concentration and drying, the mixture was dissolved in 20. Mu.L of methanol, and after 0.22 μm was subjected to HPLC analysis.

10. Subcellular localization experiments

(1) Taking out the agrobacteria GV3101 containing recombinant vector (pCAMBIA 1302-CYP 450) and the agrobacteria containing ER-marker and P19, streaking on LB solid medium containing 50mg/L Kan and Rif antibiotics, and inversely culturing at 28 ℃ for 1-2 days until single colony is grown;

(2) Picking single colony in 100 mu L LB liquid culture medium corresponding to antibiotics, shaking at 250rpm and culturing overnight at 28 ℃;

(3) Inoculating 6-14 μl of the bacterial liquid into 25mL LB liquid medium containing corresponding antibiotics, shake culturing at 28deg.C at 250rpm to OD ₆₀₀ About 0.8;

(4) Collecting bacterial liquid in a 50mL centrifuge tube, and centrifuging at 5,000rpm at room temperature for 10min;

(5) The supernatant was discarded, and the cells were resuspended in 25mL of sterile water and centrifuged at 5,000rpm for 10min at room temperature;

(6) The supernatant was discarded, and 10mL of buffer (50 mM MES, 2mM Na ₃ PO ₄ 100. Mu.M acetosyringone, 0.5% D-glucose, pH adjusted to 5.6) resuspended cells, and centrifuged at 5,000rpm for 10min at room temperature;

(7) The supernatant was discarded, the cells were resuspended in 5mL of buffer, and the OD of the cells was measured ₆₀₀ OD of P19 ₆₀₀ Adjust to OD of 1, gene and Marker ₆₀₀ And (5) adjusting to 1.5 for standby.

(8) According to the volume ratio P19: gene: marker=2: 1:1, preparing an infectious microbe liquid according to the volume ratio, standing for 2 hours, and injecting the lower tobacco epidermis by using a 10mL syringe.

(9) Pouring a proper amount of water into the injected tobacco, transferring the tobacco to a climatic chamber for culture, and observing after 48 hours;

(10) The injected leaf was cut to a size of about 0.5cm by 0.5cm with scissors, and then fluorescent signals were observed on a Leica TCS SP5 confocal laser microscope with a cover glass carrier, and photographs were taken. The excitation wavelength was set to be 488nm, the emission wavelength of GFP was set to be 490-560nm, the excitation wavelength of mCherry was set to be 552nm, and the emission wavelength of mCherry was set to be 585-680nm.

11. Transformation of transgenic hairy roots of soybean

The stored Agrobacterium rhizogenes containing the vector transformants (pK 7WG2D-CYP450, pK7 GWIHWG 2 II 2D-CYP450 and the corresponding empty vector) were taken out from the-80℃refrigerator, and a small amount of the cells were inoculated onto LB solid medium containing 50mg/L Spec and Strep by an inoculating loop, and cultured upside down at 28℃for 1-2 days.

(2) Single colonies were picked from the plates and inoculated into 5mL of LB liquid medium containing the corresponding antibiotics, and cultured at a constant temperature of 28℃for 12-16h at 200 rpm.

(3) Sucking 1mL of activated bacterial liquid, inoculating into 100mL of LB liquid medium containing corresponding antibiotics, culturing at constant temperature of 28 ℃ at 200rpm for 6-8h, and standing for OD ₆₀₀ When the value is 0.5-0.6, centrifuging at 5000rpm for 10min, removing supernatant, collecting bacterial precipitate, and waiting for infection.

(4) The germinated soybean seeds were left until the hypocotyl elongated and the two cotyledons turned green. Taking undamaged cotyledons, dividing the cotyledons into two parts, cutting off bent parts of hypocotyls, and cutting the cotyledons with a blade to form wounds with a distance of about 2cm for infection.

(5) Resuspension of the collected bacterial precipitate with 1/10B5 liquid culture medium, and adjusting bacterial liquid to OD ₆₀₀ 0.6, adding into the cut soybean cotyledons, infecting for 15min, pouring out bacterial liquid, transferring the infected soybean cotyledons onto a culture dish with sterile filter paper, and airing the water.

(6) The air-dried, infested soybean explants were facing downward and placed uniformly in plates containing 1/10B5 solid medium of moist filter paper, with approximately 20 explants placed per plate.

(7) The petri dish was sealed with a sealing film and co-cultured in the dark for 3 days.

(8) The cultured soybean explants were transferred to sterile 150mL Erlenmeyer flasks and washed 3 times with sterile water.

(9) Soaking with water added with Carb (500 mg/L) for 3h, washing with sterile water for 3 times, transferring to a culture dish with 3 layers of sterile filter paper, and air drying.

(10) The dried soybean explants were transferred to B5 solid screening solid medium supplemented with 250mg/L Carb and 5mg/L Kan corresponding plant resistance antibiotics for cultivation. After about 2 weeks, hairy roots began to appear at the wound site of the explant.

(11) After the hairy roots growing vigorously in the B5 screening solid culture medium, transferring the single plant to the B5 screening solid culture medium for culture, and waiting for the next experiment. After the hairy roots grow out, placing the hairy roots transferred into the CYP450-pK7WG2D/pK7 GWIGAG 2 II 2D corresponding vector under a fluorescence microscope (under a GFP fluorescence mode) to select out bright green roots as positive transgenic hairy roots, and then continuously culturing the transgenic hairy roots and carrying out subsequent experiments.

12. Screening of transgenic hairy roots of soybean

And (5) extracting RNA from the screened positive hairy roots by taking a proper amount of materials. Determining the purity and content of RNA, reverse transcribing the RNA into cDNA, determining the concentration of cDNA, treating H with DEPC ₂ O uniformly adjusts the cDNA concentration to 100 ng/. Mu.L, the mostThen qRT-PCR reaction is carried out. 13. Analysis of flavonoids in soy hairy roots

(1) Hairy roots grown on B5 medium for 2 weeks were harvested, fresh tissue samples were taken, ground in a mortar with liquid nitrogen and lyophilized for 2 days. Accurately weigh 20mg of sample and add 1mL of 80% methanol solution overnight. The supernatant was filtered with a 0.22 μm filter membrane after 1h of treatment with an ultrasonic apparatus (rated power, 100W) and centrifugation at 12,000rpm for 5min, and the sample was subjected to detection of total flavone content and analysis of flavonoid components in transgenic hairy roots by UPLC as follows.

(2) And determining the total flavone content by adopting an aluminum nitrate chromogenic method. Under neutral or weak alkaline condition, sodium nitrite is added to reduce flavone, aluminum nitrate is added to complex the flavone and aluminum salt to form chelate, and sodium hydroxide solution is added to open loop the flavone to form 2' -hydroxy chalcone, which has red orange color and maximum absorption peak at 510 nm.

14. Analysis and identification of flavonoid Components in enzyme Activity products and transgenic hairy roots

The enzyme activity product and flavonoid components in the transgenic hairy roots are analyzed by using UPLC, the target compound is subjected to primary mass spectrometry and secondary mass spectrometry by a liquid chromatography G6400 series triple quadrupole mass spectrometer, and the target compound is identified by combining literature reports.

(1) Ultra high performance liquid chromatography elution procedure

Ultra-high performance liquid chromatography (UPLC) adopts Agilent 1290 system, and the chromatographic column is Agilent Eclipse XDB-C18 reverse column with model number: 4.6X105 mm, particle size 5. Mu.m.

Mobile phase: phase a is an inorganic phase: 0.1% formic acid in water; the B phase is an organic phase: methanol.

Elution gradient: the initial concentration is 95% of phase A and 5% of phase B; linear gradient elution, phase a decreased from 95% to 20% and phase B increased from 5% to 80% over 16 min; phase B increased from 80% to 100% for 16-18min, then maintained for 2min, then decreased from 100% to 5% for 1min, and returned to the original concentration. The flow rate was 1mL/min.

DAD detection wavelength: 254nm (flavonol), 280nm (flavones and isoflavones).

(2) Mass spectrometry conditions

Sample preparation prior to mass spectrometry: the enzyme reaction solution was extracted with 2.5 volumes of ethyl acetate, N ₂ Blow-dried, dissolved in 100. Mu.L of methanol, and the mass spectrum identification was performed by sucking 5. Mu.L of the sample.

And separating the sample by using an Agilent liquid chromatograph G6400 series triple quadrupole mass spectrometer. The chromatographic column is an Agilent Eclipse Plus C18 reverse column, and the model is: 2.1 mm. Times.100 mm, particle size 1.8 μm. The mobile phase is the same as UPLC, the initial concentration is 95% of the A phase and 5% of the B phase; linear gradient elution, phase a decreased from 95% to 40% and phase B increased from 5% to 60% in 5 min; the phase B rises from 60% to 100% for 5-10min, and then is maintained for 2min; phase B then dropped from 100% to 5% within 0.1min, returning to the original concentration. The flow rate is 0.4mL/min, and the detection wavelength is the same.

Mass spectrometry conditions: an atmospheric pressure electrospray ion source (ESI), full ion scanning, a mass spectrometer instrument is controlled by data acquisition software (Agilent MassHunter Workstation DataAcquisition), negative ion mode Negative-ion (NI) is used for mass spectrometry, the voltage is 20-40V, and the acquisition mass spectrum range is 100-1000m/z. Finally, the data were analyzed using data analysis software (Agilent MassHunter Qualitative Analysis B.07.00).

2. Functional analysis of CYP93 family candidate genes

1. Sequence analysis and cloning of CYP93 family candidate genes

The present study downloaded Gene Model Names of soybean CYP93 family members from the P450 database (https:// drnelson. Uthsc. Edu /), translated the Gene ID by Convert Gene ModelNames Tool (https:// Soybase. Org/corruspondence /) in the Soybase website, and then analyzed the sequence information of CYP93A1 (NM_ 001254257), CYP93A2 (NM_ 001254044) and CYP93A3 (NM_ 001317523) at the Phytozome website, using Glycine max Wm82.A4.V1 versions. To understand the conservation of amino acid sequences of CYP93A1, CYP93A2 and CYP93A3, the present study performed amino acid multiple sequence alignment of members of the 13 CYP93 families in soybean (see FIG. 3), and as a result, found that the N-terminal end of the CYP93 family all has a proline-rich membrane hinge region (Proline rich membrane hinge), an I-helix region involved in oxygen binding (AGxD/ET), an E-R-R triplet (E-R-triade) domain consisting of a K-helix conserved sequence (KETLR) and an LR column conserved sequence, and a heme binding domain (FXGXXXCXG).

2. In vitro enzymatic functional assays for CYP93A1, CYP93A2 and CYP93A3

CYP93A1, CYP93A2 and CYP93A3 are cloned from soybean roots, constructed on a yeast expression vector pYeDP60, and then transferred into a yeast engineering strain WAT11 for expression of recombinant proteins. The yeast engineering strain WAT11 over-expresses an NADPH cytochrome P450 reductase gene (ATR 1) of the arabidopsis, and can provide required electrons for CYP 450. Positive yeast monoclonal was identified by PCR for in vitro enzyme function analysis.

The present study was carried out by feeding substrates in vitro to yeasts, adding 10. Mu.M 3, 9-dihydroxypterocarpine, and inducing CYP93A1, CYP93A2 and CYP93A3 recombinant proteins to express in yeasts with galactose. After the substrate and the yeast expressing the recombinant protein are co-cultured for 24 hours, the substrate and the yeast expressing the recombinant protein are extracted by using ethyl acetate with equal volume, supernatant is collected by centrifugation, concentrated and dried, then the supernatant is dissolved by using methanol, and the enzyme activity product is detected and analyzed by UPLC. As a result, it was found that CYP93A1 and CYP93A2 were able to catalyze 3, 9-dihydroxypterocarpine to generate a new peak of substance, except that CYP93A2 generated two relatively distinct peaks of products, whose retention times were 9.9min and 13.1min, respectively, labeled as P1 and P2, whereas no new peak of substance was found in the enzyme activity product of CYP93A3 with 3, 9-dihydroxypterocarpine as a substrate. The product peak P1 is more obvious in the enzyme activity product of CYP93A1, and the product peak P2 is not obvious. By performing ultraviolet absorption spectrum analysis on the product peaks P1 and P2, the present study found that the ultraviolet absorption spectrum of the product peak P1 was similar to that of 3, 9-dihydroxypterocarpan (3, 9-dihydroxypterocarpan), and that of the product peak P2 was similar to that of Daidzein (FIG. 5). The present study speculates that product peak P1 may be Glycinol, while product peak P2 may be daidzein. To verify the hypothesis of this study, this study first passed the product peak P2 along with standard daidzein through UPLC analysis, and as a result, it was found that the retention time and uv absorbance profile of the product peak P2 and standard daidzein were consistent (see fig. 4 and 5).

The product peak P1 is identified by mass spectrometry, and primary and secondary mass spectrograms of the product peak P1, the product peak P2 and standard products 3, 9-dihydroxypterocarpan alkali and daidzein are obtained. As a result, it was found that the molecular ion peak [ M-H ] -of the product peak P1 in the negative ion mode was 271.0671 (FIG. 6B). Also in negative ion mode, the results of secondary mass spectrometry (MS/MS) showed that the characteristic ion fragments m/z of the product peak P1 were 135.0087, 143.0357, 149.0246, 169.0657, 177.0193, 196.0527, 211.0396, 224.0480, 239.0341, 253.0506, 271.0651 (fig. 6B), respectively, while having characteristic ion fragments of 3, 9-dihydroxypterocarpine and daidzein. Whereas the molecular ion peak [ M-H ] -of the product peak P2 in negative ion mode was 253.0541, the characteristic ion fragments M/z of the secondary mass spectrum were 132.0216, 143.0496, 155.0500, 169.0654, 180.0579, 195.0455, 208.0530, 223.0399, 253.0509, respectively, substantially conforming to the characteristic ion fragments of the standard daidzein (FIGS. 6C and D). In summary, product peak P1 is Glycinol, product peak P2 is daidzein, CYP93A1 and CYP93A2 catalyze the production of 3, 9-dihydroxypterocarpan to Glycinol and daidzein.

Since daidzein and Glycinol are located upstream and downstream of 3, 9-dihydroxypterocarpan, respectively, in the currently predicted metabolic biosynthetic pathway of soybean isoflavone, this study hypothesized that CYP93A1 and CYP93A2 also catalyze the production of downstream products of daidzein, and as a result, the present study fed yeast strains that heterologously expressed CYP93A1 and CYP93A2 with daidzein as a substrate, found that CYP93A1 and CYP93A2 were not catalytically active against daidzein. Since both substrates for CYP93B and CYP93C were flavanones, two flavanones, which are representative of soy, were selected for this study: in vitro feeding of yeast strains that heterologously expressed CYP93A1 and CYP93A2 with glycyrrhizin and naringenin as substrates revealed that CYP93A1 was active only on glycyrrhizin, and that the products were identified as daidzein by UPLC and mass spectrometry (FIGS. 8, 9 and 10). Meanwhile, the three substrates are fed to the yeast strain which heterologously expresses CYP93A3, and the CYP93A3 is not active to the 4 substrates. 3. Three-dimensional structural simulation of CYP93A1, CYP93A2 and CYP93A3 proteins and molecular docking

The amino acid sequences of CYP93A1, CYP93A2 and CYP93A3 are very similar, the evolutionary relationship is relatively close, but the functions are different, in order to explore the mechanism, the high-level structure prediction of the protein is carried out through the I-TASSER website, and a protein three-dimensional structure model with homology modeling is obtained. The three-dimensional structural models of CYP93A1, CYP93A2 and CYP93A3 were found to be similar, but the number and positions of alpha-helices, beta-sheets and random coils in the protein molecule were varied.

Because the catalytic centers of CYP450 proteins are all combined by Heme (HEM) ligand which chelates Fe atoms, the three-dimensional structure model of the protein constructed together with heme is selected to be in molecular butt joint with a substrate 3, 9-dihydroxypterocarpan. Docking was performed using autodock4.0 software centering on the coordinates of Fe in the HEM, with the docking lattice sized to 40×46×40, and as a result, it was found that the location where HEM binds was 501 in CYP93A1 and CYP93A2, and 430 in CYP93A3 (see fig. 12). The amino acid residues in CYP93A1 which can form hydrogen bonds with HEM are phenylalanine at position 115 (Phe 115), lysine at position 128 (Lys 128), glycine at position 441 (Gly 441) and arginine at position 445 (Arg 445), respectively, at a distance of 2.9 、1.5/>、3.2/>And 3.6->. Aspartic acid at position 302 (Asp 302) forms a distance of 2.2 +.>And HEM can form a hydroxyl group at the 4' -position with 3, 9-dihydroxypterocarpineAt a distance of 2.0 +.>Hydrogen bond of (c) is provided. The amino acid residues in CYP93A2 which can form hydrogen bonds with HEM are arginine at position 369 (Arg 438), glycine at position 434 (Gly 434), serine at position 435 (Ser 435) and arginine at position 438 (Arg 438), respectively, the distance for forming hydrogen bonds is 2.8%>、2.4/>、2.7/>And 3.5->. Proline at position 432 (Pro 432) and glycine at position 364 (Gly 364) may form a distance of 2.4>And 2.3->HEM501 can form a 2.6 +.with the 4' -hydroxyl group of 3, 9-dihydroxypterocarpine>Hydrogen bonds of (e.g., table 4). The amino acid residues in CYP93A3 which can form hydrogen bonds with HEM are phenylalanine 441 (Phe 441) and serine 443 (Ser 443), respectively, at a distance of 3.2>And 2.9->. Isoglutamine at position 306The alanine at position 307 (Ala 307) and aspartic acid at position 310 (Asp 310) of the acid (Ile 306) can form a distance of 2.2 respectively from the hydroxyl group at position 7 of 3, 9-dihydroxypterocarpine>、2.4/>And 2.5->Leucine (Leu 374) at position 374 can form a distance of 2.2 +. >And HEM 430 is unable to form hydrogen bonds with 3, 9-dihydroxypterocarpine (see table 4).

TABLE 4 amino acid residues of 3, 9-dihydroxypterocarpine and HEM in combination with CYP93A1, CYP93A2 and CYP93A3 structural models

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4. Subcellular localization of CYP93A1 and CYP93A2

To understand the localization of CYP93A1 and CYP93A2 in cells, this experiment constructs CYP93A1 and CYP93A2 onto pCAMBIA1302-GFP vectors, respectively. They were then transiently expressed in tobacco leaves, respectively, and after 48 hours were observed by confocal microscopy. As a result, it was found that the green fluorescent signals of the fusion proteins CYP93A1-GFP and CYP93A2-GFP all overlapped with the mCherry fluorescent signal of the endoplasmic reticulum Marker, while the pCAMBIA1302-GFP fluorescent signal exhibited a typical whole cell distribution pattern, indicating that both fusion proteins CYP93A1-GFP and CYP93A2-GFP were localized on the endoplasmic reticulum (FIG. 13).

5. Tissue expression profiling of CYP93A1, CYP93A2 and CYP93A3

Gene expression of CYP93A1, CYP93A2 and CYP93A3 at different tissues and seed development stages of soybean including roots, stems, leaves, flowers, pods 10 days after flowering, seeds 20 days after flowering were analyzed by qRT-PCR (see fig. 14). CYP93A1 was found to be expressed in all of these 6 developmental stages of tissue, with the highest expression level in the root, lowest expression level in the stem, and relatively high expression level of CYP93A2 in the root and leaf, followed by 20 days post-flowering seed and 10 days pod, with the lowest expression level in the stem and flower, and with the highest expression level of CYP93A3 in the root, followed by leaves and stems, with little expression in the flower, 10 days post-flowering pod, and 20 days seed.

6. Induction and screening of CYP93A1 and CYP93A2 transgenic hairy roots

In order to investigate the function of CYP93A1 and CYP93A2 in soybean, the present study constructed CYP93A1 and CYP93A2 into plant overexpression vector pK7WG2D and RNAi vector pK7GWIWG2 body screening, respectively, and transferred into agrobacterium rhizogenes ARqual. The germinated soybean cotyledons are selected as explants for infection, and the induced hairy roots are selected under a fluorescence microscope to generate green fluorescence transgenic positive hairy roots (as shown in figure 15). Subsequently, RNA was extracted, reverse transcribed into cDNA, and hairy roots with high or low expression were selected by qRT-PCR. Hairy roots OE-145 and OE-156 over-expressed by CYP93A1 are screened at present, and RNAi-67, RNAi-93 and RNAi-201 (shown in figure 16A) with low expression are selected; hairy roots OE-89 and OE-108 overexpressed by CYP93A2, RNAi-16 and RNAi-67 underexpressed (see FIG. 16).

7. Flavone analysis of CYP93A1 and CYP93A2 transgenic hairy roots

The present study first analyzed the total flavonoids in CYP93A1 and CYP93A2 transgenic hairy roots, and found that there was no significant difference between the total flavonoids content in transgenic hairy roots with reduced expression levels, whether over-expressed or under-expressed, and the control (see FIG. 17).

Subsequently, the present study analyzed isoflavone compounds in CYP93A1 and CYP93A2 transgenic hairy roots by HPLC, and found that the isoflavone change trend in CYP93A1 and CYP93A2 transgenic hairy roots is basically the same, the content of daidzein and genistein is reduced compared with that of the overexpressed hairy roots and controls, and the content of daidzein is almost not different compared with that of the low-expression hairy roots and controls, but the content of genistein is obviously increased. Wherein the daidzein content in OE-145 and OE-156 of CYP93A1 is reduced by 42.93% and 38.57%, respectively, as compared to the control, and the daidzein content in OE-89 and OE-108 of CYP93A2 is reduced by 29.4% and 52.53%, respectively, as compared to the control. The genistein content in RNAi-67, RNAi-93 and RNAi-201 of CYP93A1 was increased by 135.96%, 132.96% and 81.1% respectively compared to the control; the genistein content in RNAi-16 and RNAi-67 of CYP93A2 was increased by 72.5% and 114.97%, respectively, compared to the control (FIG. 18).

3. Functional analysis of CYP82 family candidate genes

1. Cloning and sequence analysis of CYP82 family candidate genes

The study was run from the P450 database (https:/(drnelson.uthsc.edu /) download Gene Model Names of soybean CYP82 family members, through Convert Gene ModelNames tool in the soybase website (https: the Gene ID was converted by/(soybase. Org/corresponse /), and then the sequence information of CYP82A2 (nm_ 001253148), CYP82A3 (nm_ 001254043), CYP82A4 (nm_ 001317501), CYP82a23 (xm_ 003554026), CYP82C20 (xm_ 006573552) and CYP82D26 (xm_ 003528163) was analyzed on the phytozome website by using the Glycine max wm82.a4.v1 version.

2. In vitro enzyme activity assay of recombinant proteins of CYP82 family candidate genes

The study selected 10 representative flavonoid compounds including flavanones; dihydromyricetin (dihydroomyricetin), eriodictyol (Eriodictyol), liquiritin (Liquiritigenin), naringenin (Naringenin); flavone: apigenin (Apigenin); flavonols: kaempferol (Kaempferol); isoflavone: daidzein, genistein, 2' -hydroxy Genistein and 3, 9-dihydroxypterocarpan are used as substrates, and yeast strains which heterologously express CYP82 candidate genes in yeast are fed for 24 hours in vitro, extracted with an equal volume of ethyl acetate, concentrated and dried, dissolved with methanol, and the yeast feeding products are analyzed by UPLC. As a result, only CYP82D26 can catalyze naringenin to generate a new substance peak (shown in figure 19), and the graph time and the ultraviolet absorption spectrum of the product peak and the daidzein are consistent (shown in figure 20) through comparison of standards. The product peaks were then analyzed by MS and MS/MS to find that they both match the mass spectrum of daidzein (see figure 21), i.e., CYP82D26 catalyzes the production of daidzein from naringenin with a conversion of 6.02% (see figure 22).

3. Subcellular localization of CYP82D26

To understand the localization of CYP82D26 in cells, this experiment was constructed on pCAMBIA1302-GFP vector, respectively. It was then transiently expressed in tobacco leaves and observed by confocal microscopy after 48 hours. As a result, it was found that the green fluorescent signal of the CYP82D26 fusion protein and the mCherry fluorescent signal of the endoplasmic reticulum marker overlap each other, while the pCAMBIA1302-GFP fluorescent signal exhibited a typical whole cell distribution pattern, indicating that CYP82D26 was localized on the endoplasmic reticulum (see FIG. 23).

4. Analysis of expression profile of CYP82D26

Gene expression of CYP82D26 at different tissue and seed development stages of soybean including roots, stems, leaves, flowers, pods 10 days after flowering, seeds 20 days after flowering were analyzed by qRT-PCR (see fig. 24). CYP82D26 was found to be expressed most in roots, and secondly in stems and leaves, and least in seeds 20 days after flowering.

5. Three-dimensional structure simulation and molecular docking of CYP82D26 protein

In order to explore the mechanism of CYP82D26 in catalyzing naringenin, a homology modeling protein three-dimensional structure model was obtained through the I-TASSER website. Docking was performed using autodock4.0 software, centered on the Fe coordinates in the HEM, with the docking lattice sized 40 as the center, and as a result, it was found that arginine (Arg 476) at positions 130 and 476 of CYP82D26 could form distances of 1.4 from the HEM, respectively And 2.9->Valine (Val 406) at position 406 can form a distance of 3.1 +.>Hydrogen bond of (c) is provided. Tyrosine at position 141 of CYP82D26 (Tyr 141) can form a distance of 2.8 +.>Is 2.9 × distant from the O atom in position 4 by hydrogen bond>The aspartic acid at position 340 (Asp 340) can form a distance of 2.1 +.>Arginine at position 476 (Arg 476) forms 2.1 +.with the hydroxy group at position 4 of naringenin>Is 3.2 +.f from the hydroxyl group at the 4 naringenin position>Hydrogen bonds of (e.g., fig. 25 and table 4).

TABLE 4 amino acid residues of naringenin and HEM in combination with CYP82D26 structural model

6. Induction and screening of CYP82D26 transgenic hairy roots

To investigate the function of CYP82D26 in soybean, the present study constructed CYP82D26 into plant overexpression vector pK7WG2D and RNAi vector pK7 gwwg 2 in vivo studies, transferred into agrobacterium rhizogenes ARqual rods. The germinated soybean cotyledon is selected as an explant for infection, and the induced hairy roots are selected to generate green fluorescence transgenic positive hairy roots under a fluorescence microscope. Subsequently, RNA was extracted, reverse transcribed into cDNA, and hairy roots with high or low expression were selected by qRT-PCR. Hairy roots OE-82 and OE-83 overexpressed by CYP82D26 were currently screened for, and RNAi-24, RNAi-25 and RNAi-64 were underexpressed (FIG. 26).

7. Flavone analysis of CYP82D26 transgenic hairy roots

The present study first analyzed the total flavonoids in CYP82D26 transgenic hairy roots, and found that there was no significant difference between the total flavonoids content in transgenic hairy roots with reduced expression level and control, whether over-expressed or not (FIG. 27A). Subsequently, the present study analyzed isoflavone compounds in CYP82D26 transgenic hairy roots by HPLC, and found that the content of daidzein is reduced in hairy roots with reduced over-expression and expression level compared with control; while the content of genistein is reduced in the transgenic hairy roots overexpressed and increased in the transgenic hairy roots whose expression level is reduced. Wherein the daidzein content in OE-82 and OE-83 of CYP82D26 is reduced by 34.11% and 20.86%, respectively, and RNAi-24, RNAi-25 and RNAi-64 are reduced by 23.13%,15.07% and 20.71%, respectively, as compared to the control. The genistein content in OE-82 and OE-83 of CYP82D26 is reduced by 27.16% and 21.7% respectively compared with the control; the genistein content in RNAi-24, RNAi-25 and RNAi-64 was increased by 11.62%,62.78% and 15.40%, respectively.

In conclusion, by feeding yeast expressing recombinant proteins in vitro, it was found that CYP93A1 and CYP93A2 not only have P6. Alpha.H activity, catalyzing 3, 9-dihydroxypterocarpine to generate Glycinol, but also CYP93A1 and CYP93A2 can catalyze 3, 9-dihydroxypterocarpine to generate daidzein. CYP92A1 catalyzes the production of 3, 9-dihydroxypterocarpine primarily as Glycinol, whereas CYP92A2 produces less Glycinol than CYP93A1, but with significantly higher efficiency than CYP93A1. Although the evolution relationship between CYP93A3 and CYP93A1 and CYP93A2 is relatively close, and the amino acid sequence similarity is very high, the research result shows that the CYP93A3 has no catalytic activity on 3, 9-dihydroxypterocarpine. The CYP93A2 has higher specificity of catalytic substrate and only has catalytic activity on 3, 9-dihydroxypterocarpine, while CYP93A1 can catalyze 3, 9-dihydroxypterocarpine to generate Glycinol and daidzein, and can catalyze glycyrrhizin to generate daidzein, which shows that the CYP93A2 also has weak activity of iso Huang Tongmei, which is consistent with the result in the evolution analysis, and provides molecular evidence for the CYP93C to evolve into CYP93A from the aspect of in vitro enzyme function.

By constructing 6 candidate genes in the CYP82 family into the yeast expression vector pYeDP60 and transforming into the yeast strain WAT 11. As a result of feeding yeast expressing the recombinant protein with a substrate, it was found that 10 representative flavonoid compounds in an attempt, including: dihydromyricetin, eriodictyol, glycyrrhizin, naringenin, apigenin, kaempferol, daidzein, genistein, 2-substituted hydroxy genistein and 3, 9-dihydroxypterocarpine are used as substrates, and only CYP82D26 can catalyze naringenin to generate daidzein.

CYP93A1, CYP93A2 and CYP82D26 are all endoplasmic reticulum localization, the three are expressed in various tissues, the CYP93A1 and CYP82D26 are the highest in the root, the CYP93A2 is expressed in the root and the leaf, the isoflavone compound in the soybean root is mainly daidzein, the glycoside daidzein and malonyl daidzein, and the CYP93A1, the CYP93A2 and the CYP82D26 probably play an important role in the biosynthesis process of daidzein. CYP93A3 is also the gene whose expression level is highest in roots, but it is inactive and may be functionally redundant.

The total flavone content in transgenic hairy roots with over-expression and reduced expression levels of CYP93A1, CYP93A2 and CYP82D26 are not obviously different from that of the control, but the content of daidzein and genistein is changed. Genistein in the transgenic hairy roots overexpressed by CYP93A1, CYP93A2 and CYP82D26 was reduced as compared to the control, while the content of genistein in the transgenic hairy roots whose expression amount was reduced was increased as compared to the control. The in vitro enzyme-linked results of this study showed that CYP93A1, CYP93A2 and CYP82D26 are all involved in the synthesis of daidzein, which has a substrate competing relationship with genistein. The reduced levels of daidzein in the transgenic hairy roots over-expressed by CYP93A1 and CYP93A2 compared to the control, while no significant difference in daidzein levels in the hairy roots with low expression levels compared to the control, indicated that CYP93A1 and CYP93A2 were located mainly downstream of daidzein in the soybean body. The in vitro enzyme activity results of this study also showed that CYP93A1 and CYP93A2 catalyzed the conversion efficiency of 3, 9-dihydroxypterocarpan to Glycinol was higher. The reduced daidzein content in transgenic hairy roots with reduced CYP82D26 overexpression and expression suggests that natural substrates for CYP82D26 in soybean may not be only naringenin, but may also catalyze compounds downstream of daidzein, and in vitro enzyme activity results of this study also found that CYP82D26 catalyzes naringenin to form daidzein.

The application of the CYP450 gene provided by the invention in regulating and controlling the synthesis of soybean isoflavone is described in detail. The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.

Claims

Application of CYP82D26 gene in regulating and controlling soybean isoflavone synthesis.
2. The use according to claim 1, wherein the CYP82D26 gene is endoplasmic reticulum localization.
3. The use according to claim 1, wherein the protein encoded by the CYP82D26 gene is catalytically active against naringenin and catalyzes the production of daidzein from naringenin.
Application of CYP82D26 gene in regulating and controlling daidzein synthesis.
Use of the cyp82d26 gene in the cultivation of a transgenic plant, wherein said transgenic plant produces soy isoflavones.
6. The use according to claim 5, wherein the natural substrate catalyzed by the CYP82D26 gene encodes a protein comprising naringenin and a compound downstream of daidzein.