CN111690662B

CN111690662B - Application of soybean bHLH transcription factor GmPIF1 gene in promotion of isoflavone synthesis

Info

Publication number: CN111690662B
Application number: CN202010497098.1A
Authority: CN
Inventors: 王庆钰; 王天亮; 刘雅婧; 王英; 李景文; 闫帆; 王硕; 张鑫生; 杨旭光
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2020-06-03
Filing date: 2020-06-03
Publication date: 2021-11-12
Anticipated expiration: 2040-06-03
Also published as: CN111690662A

Abstract

The invention relates to an application of soybean bHLH transcription factor GmPIF1 gene in promoting isoflavone synthesis, belonging to the fields of genetic engineering and molecular biology. The soybean bHLH transcription factor GmPIF1 gene is applied to promotion of isoflavone synthesis, and the nucleotide sequence of the soybean bHLH transcription factor GmPIF1 gene is shown in SEQ ID No. 1. The beneficial effects are that: the GmPIF1 promotes the accumulation of isoflavone in the soybean hairy roots, provides a new method for improving the content of the isoflavone, can synthesize the isoflavone with higher efficiency by utilizing a biological engineering technology and a gene regulation method, and overcomes the defects of long artificial cultivation and breeding period, unstable hybridization character, difficult screening and the like. The transcription factor GmPIF1 gene is transferred into soybean hairy roots to cause the soybean hairy roots to be over-expressed, so that the content of isoflavone in the hairy roots is increased, and the expression of related enzyme genes in an isoflavone synthesis path is regulated.

Description

Application of soybean bHLH transcription factor GmPIF1 gene in promotion of isoflavone synthesis

Technical Field

The invention relates to the field of genetic engineering and molecular biology, in particular to application of a red light and far-red light mediated transcription factor gene GmPIF1 in soybean in promoting isoflavone synthesis.

Background

Soybean (Glycine max) is an important economic crop, can provide metabolic products such as vegetable oil, vegetable protein, isoflavone and the like for human beings, wherein the soybean isoflavone has important commodity value as a health product, and has good effects on reducing the morbidity of cardiovascular diseases and hyperlipidemia, preventing breast diseases and osteoporosis, resisting oxidation, relieving climacteric syndrome, resisting tumors and the like. In order to meet the market demand, the cultivation of soybean varieties with high isoflavone quality is receiving wide attention of researchers.

Soy isoflavones are secondary metabolites in the phenylalanine metabolic pathway in plants, and their synthesis is influenced by a number of factors, including the environment and the catalytic enzyme gene activity in the isoflavonoid syntactical pathway. Although the optimal production environment of soybean is known, the field environment is often uncontrollable, so that the research on the influence of soybean genes on the accumulation of isoflavones becomes an important research direction. In soybeans, isoflavones accumulate to different extents in each tissue, with the greatest accumulation in roots and embryos, increasing in isoflavone content as the embryos develop, and the greatest accumulation in mature kernels. The synthesis of soybean isoflavone in vivo is regulated by multiple catalytic enzymes and transcription factors.

Phenylalanine metabolism is initiated by Phenylalanine Ammonia Lyase (PAL), cinnamic acid 4-hydroxylase (C4H) and coumarate-coa ligase (4CL) with a series of enzymatic transformations, the metabolic pathways of which have been extensively studied over the past few decades. Isoflavone synthesis begins with the leguminous specific branch of the phenylalanine pathway catalyzed by isoflavone synthase/chalcone isomerase (IFS/CHI). The process takes chalcone as a substrate, and three free aglycones are formed under the catalysis of chalcone reductase (CHR), CHI, isoflavone O-methyltransferase (IOMT) and isoflavone synthase (IFS): daidzein, glycitein, and genistein. The aglycones of the glycoside are catalyzed by isoflavone-7-glycosyltransferase (IF7GT) to form bound isoflavones: daidzin, glycitin, and genistin. Uridine diphosphate-dependent glycosyltransferases (UGTs) and malonyl-coa-dependent acyltransferases (MATs) catalyze the modification step of soy isoflavone aglycones. Daidzein, genistein, daidzein, and their malonyl forms accumulate to varying degrees in various tissues and organs.

The expression of the catalytic enzyme gene in the phenylalanine metabolic pathway is regulated by transcription factors. To date, much research has focused on key enzymes in metabolic pathways, and research on transcription factors that regulate isoflavone synthesis has been limited. Therefore, it is especially important to deeply study the transcription factor regulating the synthesis of isoflavone and the molecular mechanism of its action in order to obtain the soybean of high isoflavone variety. In plant growth and development and response to the external environment, transcription factors regulate gene transcription levels to determine many important physiological processes and phenotypic changes. Studies have shown that bHLH transcription factors are one of the largest transcription factor families involved almost throughout plant growth and development, including: environmental factor responses, cell elongation, metabolism of secondary products, and regulation of phenylalanine metabolic pathways in various species. Phenylalanine metabolic pathways are ubiquitous in plants, and metabolites are a series of flavonoids including flavones, anthocyanins, isoflavones, phytoalexins and the like. The PIF class of transcription factors belongs to the 24 subfamily of bHLH transcription factors. All PIF proteins contain an APB (Active Phytochrome B-binding) domain that binds to PHYB. Wherein AtPIF1 and AtPIF3 not only have APB domain, but also contain an APA domain that binds PHYA. In arabidopsis thaliana, AtPIF1 was shown to be involved in anthocyanin synthesis. The PIF transcription factor has the functions of promoting the elongation of hypocotyl, inhibiting the expansion of cotyledon, keeping the top hook, promoting the synthesis of anthocyanin, inhibiting the germination of seeds, inhibiting the development of chloroplast and the like. In Arabidopsis, PIF3 can be combined with a promoter of an anthocyanin synthesis gene to promote synthesis of anthocyanin and induce transcription of related synthetase.

So far, no report is found on the research of soybean GmPIF1 gene. Therefore, the cloning and function research of the GmPIF1 gene in the soybean are of great significance.

Disclosure of Invention

The invention provides application of a soybean bHLH transcription factor GmPIF1 gene in promoting isoflavone synthesis, which aims to clone a transcription factor gene GmPIF1 for regulating and controlling the synthesis of soybean isoflavone from soybeans, and defines the application of the transcription factor, namely application of GmPIF1 in improving the content of total isoflavone in soybean hairy roots and increasing the transcription level of catalytic enzyme in an isoflavone synthesis path.

The technical scheme adopted by the invention is as follows:

an application of soybean bHLH transcription factor GmPIF1 gene in promoting isoflavone synthesis.

The nucleotide sequence of the soybean bHLH transcription factor GmPIF1 gene is shown in SEQ ID No. 1.

The amino acid sequence of the soybean bHLH transcription factor GmPIF1 gene coding protein is shown in SEQ ID No. 2.

The GmPIF1 gene promotes the accumulation of isoflavone in soybean hairy roots.

The transcription factor GmPIF1 gene is transferred into soybean hairy roots to regulate and control the expression of related enzyme genes in an isoflavone synthesis pathway.

The invention selects a bHLH transcription factor gene with the transcription level improved along with the development increase of soybean embryo from the digital expression profiles of the genes of different periods of soybean Jilin 32 grains, clones the gene from the soybean Jilin 32, and then carries out functional identification on the encoded protein. The transcription factor gene is named GmPIF1 through amino acid sequence and family evolutionary tree analysis. The cDNA size of the transcription factor is 1530bp, the full-length sequence of the transcription factor is shown as a sequence SEQ ID No.1, and 509 amino acid proteins are coded and are shown as a sequence SEQ ID No. 2. The CDS full-length sequence of GmPIF1 is inserted into a pHB-YFP plant expression vector to construct an over-expressed recombinant vector pHB-GmPIF1-YFP, part of CDS of GmPIF1 is inserted into a pFGC5941 vector to construct an RNAi interference vector pFGC5941-GmPIF1, and the plant over-expression and RNAi interference vector is transferred into the rooting of soybean Jilin 32 by utilizing an agrobacterium-mediated method, so that the accumulation of soybean isoflavone can be improved, and the expression of related enzyme genes in a phenylalanine metabolic pathway can be regulated.

The invention has the beneficial effects that: the invention clones a soybean GmPIF1 transcription factor gene related to a soybean phenylalanine metabolic pathway, obtains a homologous gene of the transcription factor through evolutionary analysis, and learns that the transcription factor belongs to a bHLH gene family; further, soybean hairy roots are transformed by the constructed recombinant plant expression vectors of pHB-GmPIF1-YFP and pFGC5941-GmPIF1, and the isoflavone content of the positive hairy roots is detected, so that the GmPIF1 promotes the accumulation of isoflavone in the soybean hairy roots. The invention provides a new method for improving the content of soybean isoflavone, can synthesize the soybean isoflavone with higher efficiency by utilizing a biological engineering technology and a gene regulation method, and overcomes the defects of long artificial cultivation breeding period, unstable hybridization character, difficult screening and the like. The transcription factor GmPIF1 gene is transferred into soybean hairy roots to cause the soybean hairy roots to be over-expressed, so that the content of isoflavone in the hairy roots is increased, the expression of related enzyme genes in an isoflavone synthesis path is regulated, and the results provide theoretical reference and scientific basis for large-scale industrialized production of isoflavone.

Drawings

FIG. 1 is a graph of the PCR amplification results of GmPIF1, in which M:2000bp maker.1: GmPIF 1;

FIG. 2 is a graph of a phylogenetic tree analysis of GmPIF1 and AtPIFs proteins;

FIG. 3 is an alignment of the amino acid sequences of GmPIF1 and AtPIF1, with APB, APA, bHLH in bold lines representing predicted corresponding domain amino acid residues and arrows representing key amino acid residues for AtPIF1 to interact with AtPHYB and AtPHYA, respectively, in the putative predicted APB and APA motifs;

FIG. 4 is a tissue-specific expression map of GmPIF1 gene;

FIG. 5 is a subcellular localization of GmPIF1, 35S: YFP denotes pHB-YFP transduced tobacco cells; 35S, GmPIF1, YFP indicates pHB-GmPIF1-YFP transduced tobacco cells;

FIG. 6 is a prokaryotic expression diagram of GmPIF1, M. Low molecular weight protein Marker,1.pET-28a (+) induced for 6 h; 2-9.pET28a-GmPIF1 induces 0h, 1h, 2h, 3h, 4h and 6 h;

FIG. 7 is a graph showing the verification of the transcriptional self-activation activity of GmPIF1 protein in a yeast system, wherein BK/AD, pGBKT7 and pGADT7 co-transformed yeast are used as negative controls; BK-53/AD-T, pGBKT7-53 and pGADT7-T co-transformed yeast as positive controls; GmPIF1/AD, pGBKT7-GmPIF1 and pGADT7 co-transformed yeast;

FIG. 8 is a YFP fluorescence validation graph of soybean hairy roots over-expressed with GmPIF1, wherein Control is non-transgenic hairy roots; the GmPIF1-YFP-1, GmPIF1-YFP-2 and GmPIF1-YFP-3 are 3 pHB-GmPIF1-YFP transgenic soybean hairy roots;

fig. 9 is a PCR validation graph of GmPIF1 RNAi interfering with soybean hairy roots, in which:

+. pFGC5941-GmPIF1 plasmid; non-transgenic hairy roots; m.5000marker; pFGC5941-GmPIF1 transgenic soybean hairy root

FIG. 10 is a graph showing the expression levels of enzymes involved in the phenylalanine metabolic pathway in transgenic hairy roots, Control being non-transgenic hairy roots; the GmPIF1-OE is GmPIF1 overexpression hairy roots; GmPIF1-RNAi is GmPIF1 RNAi interference hair roots. Asterisks represent significance of differences from non-transgenes in the corresponding group as tested against t (;, P < 0.01;, P < 0.05);

FIG. 11 is a graph of the results of HPLC measurements of isoflavone content in transgenic hairy roots, with Control being non-transgenic hairy roots; the GmPIF1-OE is GmPIF1 overexpression hairy roots; GmPIF1-RNAi is GmPIF1 RNAi interference hair roots. Asterisks represent significance of differences from non-transgenic genes in the corresponding group as tested against t (;, P < 0.01;, P < 0.05).

Detailed Description

The present invention is further illustrated by the following figures and examples, which are not intended to limit the scope of the invention, wherein the examples are intended to be exemplary of conventional methods unless otherwise specified, and wherein the reagents used are, unless otherwise specified, either conventional commercial reagents or reagents formulated according to conventional methods.

Example 1: cloning of GmPIF1 transcription factor gene and biological analysis thereof

Screening a group of genes with the increasing expression level along with the development of soybean embryos from constructed digital expression profiles of genes of different periods of soybean Jilin 32 grains, wherein the genes are considered to have the function of potentially regulating the synthesis of soybean isoflavone, and GmPIF1 is one of bHLH transcription factors. The total RNA of the soybean variety Jilin 32 leaf was extracted from 40mg of soybean Jilin 32 three-leaf complex by RNAlso Reagent (purchased from TaKaRa), and the integrity of the RNA was checked by 1% agarose electrophoresis. The cDNA was synthesized according to the instructions for Reverse Transcriptase M-MLV (RNase H-).

According to the CDS sequence of GmPIF1 in NCBI, a primer sequence is designed, soybean Jilin 32 leaf cDNA is used as a template, and a reverse transcription PCR (RT-PCR) technology is utilized to obtain a PCR product of the CDS full length of GmPIF 1.

GmPIF1 (accession number: XP-006594070) cloning primer:

GmPIF1–F：5’-ATGAATCACTGTGTTCCAGATTTCG-3’

GmPIF1–R：5’-TCATGTTTCGTCTGACTGATGCTTT-3’

the PCR reaction system is as follows:

the PCR reaction conditions are as follows:

the PCR product was subjected to agarose gel electrophoresis, the desired fragment was recovered by cutting the gel, and the recovered fragment was ligated with the intermediate vector pMD18-T (purchased from TaKaRa Co.). Transferring the connecting liquid into Escherichia coli (E.coli) DH5 alpha to be competent, screening and culturing on LB solid culture medium containing ampicillin, selecting monoclonal and carrying out PCR detection on bacterial liquid, and sequencing the positive monoclonal bacterial liquid. And preserving bacteria of the bacterial liquid with correct sequencing and preparing the quality-improved particles (pMD18-T-GmPIF1) for later use.

GmPIF1 and its homologous sequences are all from NCBI database (https://www.ncbi.nlm.nih.gov/) (ii) a Using ExPASy (A), (B), (C), (D) and D) ahttps://web.expasy.org/compute_pi/) Predicting the molecular weight and isoelectric point of the protein; using DNMAN v.7 and Clustal W2(https://www.ebi.ac.uk/Tools/msa/clustalw2/) Performing multiple sequence alignment; the evolutionary tree analysis was performed using MEGA 6.0.

The 1530bp GmPIF1 gene full-length CDS sequence (shown in figure 1) is obtained by PCR amplification, the nucleotide sequence of the CDS sequence is shown as SEQ ID No.1, a protein consisting of 509 amino acid residues is coded, the amino acid sequence is shown as SEQ ID No.2, the molecular mass of the protein is 56.36kDa, and the isoelectric point is 5.68. Evolutionary tree analysis revealed that GmPIF1 is closely related to arabidopsis thaliana AtPIF1 (fig. 2), belongs to the PIF subfamily of bHLH transcription factors, and comprises a conserved HLH binding domain, an APA domain and an APB domain (fig. 3).

Example 2: tissue-specific expression analysis of GmPIF1 Gene

Total RNAs of roots, stems, leaves, flowers, 20-day embryos, 30-day embryos and 50-day embryos of soybean variety Jilin 32 were extracted and reverse-transcribed into cDNAs in the same manner as in example 1. The expression of the GmPIF1 gene in different tissues of soybean was detected by relative fluorescent quantitative PCR (qPCR) according to SYBR O R Premix Ex Taq^TMII (from TaKaRa) instructions, using 7500 real-time fluorescence quantitative amplification instrument.

Soybean beta-tubulin is used as an internal reference gene, and the primer sequence is as follows:

tubulin-F:5’-GGAAGGCTTTCTTGCATTGGTA-3’

tubulin-R:5’-AGTGGCATCCTGGTACTGCA-3’

the quantitative primers of the GmPIF1 gene are as follows:

GmPIF1-q-F：5’-AAACCCAAATGGACGACGACTTA-3’

GmPIF1-q-R：5’-TGGCCGTTTTGCCATAACAGTTC-3’

the PCR reaction system is as follows:

the PCR reaction conditions are as follows:

data analysis used GraphPad software. The result shows that the GmPIF1 gene has the highest expression quantity in the leaves and flowers of soybeans, which is probably that GmPIF1 participates in light regulation; the expression of the GmPIF1 gene is increased along with the gradual development and maturation of soybean young embryos (figure 4), which is consistent with the trend in the gene digital expression profiles of different periods of soybean Jilin 32 grains. The expression mode of the embryo of the GmPIF1 gene in soybean development is basically consistent with the accumulation rule of soybean isoflavone in various development stages of soybean researched by predecessors, which indicates that the GmPIF1 gene may participate in the regulation and control function in the soybean isoflavone synthesis pathway.

Example 3: subcellular localization of the Soybean GmPIF1 Gene

Designing primer sequences with restriction enzymes BamH1 and Spe1 according to CDS sequence of GmPIF1, and obtaining GmPIF1 sequence containing enzyme cutting site through subcloning.

The primer sequence is as follows:

GmPIF1-YFP-F：5’-CGGGATCCATGAATCACTGTGTTCCA-3’

GmPIF1-YFP-R：5’-GGACTAGTTCATGTTTCGTCTGACTG-3’

according to the procedure of example 1, the PCR product obtained from the subcloning was ligated to the pMD18-T vector, then transformed into E.coli DH 5. alpha. and sequenced, and the correctly sequenced bacterial suspension was preserved and the plasmid was upgraded (pT-GmPIF1/BamH1/Spe1) for use. pT-GmPIF1/BamH1/Spe1 and pHB-YFP plant expression vector with CaMV 35S promoter are subjected to double enzyme digestion respectively by BamH1 and Spe1, target fragments are cut and recovered respectively, the recovered GmPIF1 fragment and the vector fragment are connected overnight, then the connecting solution is transferred into escherichia coli DH5 alpha competence, screening culture is carried out on LB solid culture medium containing kanamycin, single clone is selected and is subjected to bacteria liquid PCR detection, and positive single clone bacteria liquid is subjected to bacteria preservation and plasmid extraction (pHB-GmPIF1-YFP) for later use.

For subcellular localization, plasmids pHB-GmPIF1-YFP and pHB-YFP were transformed into Agrobacterium EHA105 and in a resuspension solution. Plasmid-containing resuspension was injected into the lower epidermis of native tobacco. After 3 days of incubation at 25 ℃ under 16h light/8 h dark photoperiod conditions, the yellow fluorescence signal was observed under a fluorescence microscope (purchased from Nikon).

Under a fluorescence microscope, yellow fluorescence signals emitted from control vector pHB-YFP (35S:: YFP) infected tobacco were observed in both nucleus and cytoplasm, while fluorescence signals emitted from pHB-GmPIF1-YFP recombinant vector (35S:: GmPIF1:: YFP) infected tobacco were limited to the nucleus (FIG. 5). This indicates that GmPIF1 is localized to the nucleus.

Example 4: expression of soybean GmPIF1 gene in pronucleus

Primers containing BamHI and Sal1 restriction enzyme sites are designed at the 5 'end and 3' end of the GmPIF1 gene respectively, and PCR amplification is carried out by taking a plasmid of pMD18-T-GmPIF1 as a template.

The primer sequence is as follows:

GmPIF1-YH-F：5’-CGGGATCCGGTAAAATGAATCACTGTGTT-3’

GmPIF1-YH-R：5’-ACGCGTCGACAACCAATCATGTTTCGTCTG-3’

according to the procedure of example 3, GmPIF1 was subcloned into pET28a (+) (purchased from Novagen) vector, and after correct identification, the recombinant plasmid pET28a (+) -GmPIF1 was transformed into Escherichia coli Rosetta (DE3), and screened and cultured on LB solid medium containing kanamycin and chloramphenicol, and a single clone was picked up and subjected to PCR detection of bacterial liquid, and a positive single clone bacterial liquid was preserved for use.

Prokaryotic expression of recombinant protein: the positive and empty vector strains were inoculated into 5mL (50. mu.g/mL kanamycin and 15. mu.g/mL chloramphenicol) LB medium, respectively, and cultured overnight at 37 ℃ at 160 rpm; inoculating to 10mL LB medium at 1%, and shake-culturing at 37 deg.C to OD₆₀₀About 0.6. IPTG (100mM) was added to a final concentration of 1.0mM, and the mixture was cultured with shaking at 37 ℃ for 1 to 6 hours. 1.5mL of induced bacteria are taken, centrifuged at 4 ℃ and 5000rpm for 5min to collect thalli, and protein is crudely extracted. SDS-PAGE analysis of expression products: the procedure was carried out according to the conventional method of SDS-PAGE electrophoresis.

Optimization of IPTG induction time shows that pET28a (+) -GmPIF1 can be induced in Rosetta (DE3), and GmPIF1 protein (about 56.36KDa) has higher expression under the induction condition of 1-6h (figure 6).

Example 5: expression of soybean GmPIF1 gene in yeast

The yeast expression vector was constructed in the same manner as in example 3. GmPIF1 was subcloned into pGBKT7 (ex clontech) vector and, when identified as correct, was expressed in yeast AH109 (ex clontech). Yeast competence preparation and transformation method reference

The Gold Yeast One-hybrid Screening System (Clontech) was performed using a manual.

pGBKT7 and pGADT7(BK/AD) were co-transferred into yeast cells as negative controls; pGBKT7-53 and pGADT7-T (BK-53/AD-T) were co-transferred into yeast cells as positive controls; after recombinant plasmids pGBKT7-GmPIF1 and pGADT7(GmPIF1/AD) are jointly transferred into yeast AH109, an experimental group and a control group can normally grow in a defective culture medium SD/-Trp/-Leu, and the expansion potential is good; however, BK/AD and GmPIF1/AD could not grow on the defective medium SD/-Trp/-Leu/-His/-Ade/(FIG. 7), indicating that GmPIF1 has no transcriptional auto-activation activity.

Example 6: agrobacterium rhizogenes-mediated transformation of soybean hairy roots

Selecting an interference fragment of the GmPIF1 gene in soybean, and designing primers of a forward fragment containing Nco1 and Asc1 enzyme cutting sites and a reverse fragment containing SmaI and BamH1 enzyme cutting sites respectively as shown in a sequence SEQ ID No.3, and carrying out PCR amplification by taking a plasmid of pMD18-T-GmPIF1 as a template.

The forward primer sequence is:

GmPIF1-RNAi-FS：5’-CATGCCATGGCGACGAGATCATGGAA-3’

GmPIF1-RNAi-RS：5’-AGGCGCGCCCCGGCGTTTCGCAAGA-3’

the reverse primer sequence is as follows:

GmPIF1-RNAi-FA：5’-TCCCCCGGGCGACGAGATCATGGAA-3’

GmPIF1-RNAi-RA：5’-CGGGATCCCCGGCGTTTCGCAAGA-3’

according to the steps in the example 2, the forward fragment and the reverse fragment are respectively subcloned into a pFGC5941 (purchased from Novagen) vector, after the correct identification, the recombinant plasmid pFGC5941-GmPIF1 with the forward fragment and the reverse fragment is transferred into escherichia coli DH5 alpha to be screened and cultured on an LB solid culture medium containing kanamycin, a single clone is selected and subjected to bacterium liquid PCR detection, and a positive single clone bacterium liquid is preserved for later use.

Bacterial liquid PCR verification primer sequence:

pFGC5941-F：5’-ATTGCGATAAAGGAAAGGCTA-3’

pFGC5941-R：5’-TATCACCCGTTACTATCGTAT-3’

plasmids carrying pHB-GmPIF1-YFP and pFGC5941-GmPIF1 are respectively transferred into agrobacterium K599 by a soybean rooting transformation method, and soybean Jilin 32 is further transformed, wherein the transformation method comprises the following steps:

(1) selecting soybean Jilin 32 with intact seed coat, no scab and no insect pest;

(2) sterilizing soybean Jilin 32 with chlorine for about 18 h;

(3) inoculating soybean seeds in a germination culture medium, and germinating for about 5 days under the conditions of 25 ℃ and 16h of illumination/8 h of darkness;

(4) preparing infection bacterial liquid: taking original bacteria liquid, streaking on solid YEP plate (adding 50mg/L Kan, 50mg/L Rif) with burnt inoculating loop, culturing at 28 deg.C for 2-3d, selecting single colony, adding into 5mL liquid YEP culture medium (adding 50mg/L Kan, 50mg/L Rif), culturing at 28 deg.C and 180rpm under shaking to OD₆₀₀About 0.8;

(5) removing seed coats of germinated soybeans, dividing cotyledons and hypocotyls into two parts along a central axis (the method is similar to cotyledonary node transformation), removing terminal buds and axillary buds, transversely cutting 3-5 knives at the joints of the cotyledonary nodes and the hypocotyls, putting into an agrobacterium infection solution, and infecting for 3 hours;

(6) putting the explant on filter paper, and sucking off the bacterial liquid on the surface of the explant;

(7) placing the explant on a co-culture medium paved with filter paper, and culturing for 5 days at 25 ℃ under 16h light/8 h dark conditions;

(8) putting the explant into a sterile 1/2MS liquid culture medium added with 250mg/L Cef and 250mg/L benzyl carboxylate, soaking and cleaning for 30min, then facing paraxial surfaces of cotyledonary nodes upwards, inserting hypocotyls into a rooting induction culture medium, and inducing the generation of rooting after 16h illumination/8 h dark culture for 10-20 days at 25 ℃;

observing a fluorescent signal through a fluorescent microscope to screen out the soybean hairy roots with transgenic positive overexpression of the GmPIF1 (figure 8); soybean hairy root DNA was extracted and PCR verified GmPIF1 RNAi interfered with transgene positive hairy roots (fig. 9). These results indicate that both the overexpression vector pHB-GmPIF1-YFP and the RNAi interference vector pFGC5941-GmPIF1 have been transformed into soybean hairy roots.

Example 7: GmPIF1 gene for regulating related gene in phenylalanine metabolic pathway

Selecting non-transgenic, over-expression and RNAi interfering soybean hairy roots which grow for about 10 days and have good growth state, respectively extracting RNA, and then carrying out reverse transcription to obtain cDNA, wherein the method is the same as the example 1. Using the cDNA as a templateDesigning key enzyme genes in the soybean phenylalanine metabolic pathway: PAL1, C4H, 4CL, CHS7, CHS8, CHR, CHI1A, CHI1B1, IFS1, IFS2, flavanone 3-hydroxylase gene (F3H) quantitative primers

TB Green^TMFast qPCR Mix kit instructions for relative fluorescent quantitative PCR.

The related primer sequences are as follows:

PAL1-F：5’-AGCAACACAACCAGGATGTCAA-3’

PAL1-R：5’-CAATTGCTTGGCAAAGTGCA-3’

C4H-F：5’-AGGCGAGATCAACGAAGACAAC-3’

C4H-R：5’-TTCACAAGCTCAGCAATGCC-3’

4CL-F：5’-AGGCAATGTACGTGGACAAGCT-3’

4CL-R：5’-TCCGAGAGGACAGAGAAGTGGA-3’

CHS7-F：5’-GCTGTGACTTGTTTTGAGTTC-3’

CHS7-R：5’-GACTTGTCTCACATGCGCTGGAA-3’

CHS8-F：5’-GCTCCCAGTACTTTAATTGATTTCTG-3’

CHS8-R：5’-GACTTGTCTCACATGCGCTGGAA-3’

CHR-F：5’-CAAAGCCATTGGAGTCAGCAA-3’

CHR-R：5’-CCATGCAAGGTTCATCTCCACT-3’

CHI1A-F：5’-GGCGCTGAATACTCAAAGAAGG-3’

CHI1A-R：5’-AGAGGCACCAGGTGCAAAATT-3’

CHI1B1-F：5’-AGCTGAATTGCTCGACTCCCT-3’

CHI1B1-R：5’-CAGATTGCATATGTGCCACACA-3’

IFS1-F：5’-AGAATTCCGTCCCGAGAGGTT-3’

IFS1-R：5’-TGCCATTCCTGAAGTAGCCAA-3’

IFS2-F：5’-AATGTGCCCTGGAGTCAATCTG-3’

IFS2-R：5’-GGCGTCACCACCCTTCAATAT-3’

F3H-F：5’-ATTCATTGTCTCCAGCCATCT-3’

F3H-R：5’-TTACTTTGTCGCTGTATTCCTCA-3’

QRT-PCR results showed that overexpression of GmPIF1 significantly increased the expression levels of PAL1, 4CL, CHI1B1, IFS2 and F3H, wherein expression levels of PAL1, 4CL and IFS2 were positively correlated with isoflavone synthesis, and CHI1B1 and F3H were involved in anthocyanin and flavone synthesis in phenylalanine metabolic pathway (fig. 10).

Example 8: influence of GmPIF1 gene overexpression on synthesis content of soybean hairy root isoflavone

Selecting about 0.5g of non-transgenic, over-expression and RNAi interference soybean hairy roots which grow for about 15 days and have good growth state, and respectively adding 80% methanol extract (400 muL 100mg per day)^-1Fresh weight ratio), grinding into slurry in a mortar, transferring the slurry into a centrifuge tube, carrying out water bath at 60 ℃ for 14h, centrifuging at 12000rpm for 15min, filtering the supernatant with a 0.45 mu m filter membrane to obtain the final pretreatment solution, and storing at 4 ℃. The isoflavone content was measured by a Japanese Jinshima high performance liquid chromatograph (LC-20A). The conditions of the high performance liquid chromatography are as follows: the chromatographic system in the high performance liquid chromatograph comprises: a DGU-20A3 degasser, 2 LC-20AT solvent array detectors, an ultraviolet detector SPD-20AV ultraviolet detector, a CTO-10AS column incubator, a CBM-20A system controller, an LC-Solution workstation and a 7725 manual sample injector; the chromatographic column is Phenomenex C18(150mm × 4.6mm, 5.0 μm); the mobile phase is methanol: water 30: 70 (v/v); flow rate: 1 mL. min^-1And the detection wavelength is as follows: 254 nm; column temperature: 40 ℃, sample introduction: 10 μ L, assay time: each sample is 40-50 min. And (3) parallelly measuring all samples for 3 times, calculating the average value of peak areas, determining the qualitative according to retention time, determining the quantitative according to the peak areas, and calculating the content of each isoflavone component in the samples according to a standard curve of a standard substance.

The results show that although there was no statistical difference between the individual isoflavone components of transgenic and non-transgenic hairy roots, the total isoflavone content in the hairy roots was significantly increased by GmPIF1 overexpression and significantly decreased by interference of GmPIF1 transcription (fig. 11).

Sequence listing

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ggcgattcga gcgggagtgc cgaaccggtc gagagagaac cgatggcgga ccggaagagg 780

aagggaaggg aacatgagga atcggagttt cagagcgagg atgttgattt tgaatctccc 840

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Asn Gln Arg Pro Phe Arg Lys Pro Pro Gln Pro Pro Glu Ala Asn Gly

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Gly Asp Gly Ala Ile Ser Ala Arg Glu Ile Arg Ser Ser Glu Ala Glu

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Asn Tyr Asn Asn Ser Gln His Leu Phe Met Gln Glu Asp Glu Met Ala

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Ala Trp Leu His Tyr Pro Ile His Glu Asp Pro Pro Pro Phe Asp His

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His Asp Phe Gly Ala Asp Ile Phe Tyr Pro Pro Pro Asn Ala Thr Ala

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Ser Gln Asn Arg Gly Ser Ala Ala Val Gln Ser Ser Phe Arg Thr Thr

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Glu Leu Trp His Pro Ala Pro Arg Pro Pro Ile Pro Pro Pro Arg Arg

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Pro Glu His Ala Pro Ser Arg Ile His Asn Phe Ala His Phe Thr Lys

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His Gly Asn Ala Ser Ser Ser Ser Lys Ala Ala Ala Ala Ala Gln Pro

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Thr Val Val Asp Ser Cys Glu Thr Pro Val Ala Thr Ala Glu His Ala

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Glu Thr Gly Arg Ala Arg Ala Ala Ala Gly Lys Thr Ala Val Ser Asp

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Gly Gly Arg Glu Thr Ala Thr Cys Asp Val Thr Val Thr Ser Ser Pro

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Gly Asp Ser Ser Gly Ser Ala Glu Pro Val Glu Arg Glu Pro Met Ala

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Asp Arg Lys Arg Lys Gly Arg Glu His Glu Glu Ser Glu Phe Gln Ser

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Glu Asp Val Asp Phe Glu Ser Pro Glu Ala Lys Lys Gln Val His Gly

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Ser Thr Ser Thr Lys Arg Ser Arg Ala Ala Glu Val His Asn Leu Ser

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Glu Arg Arg Arg Arg Asp Arg Ile Asn Glu Lys Met Lys Ala Leu Gln

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Glu Leu Ile Pro Arg Cys Asn Lys Ser Asp Lys Ala Ser Met Leu Asp

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Glu Ala Ile Glu Tyr Leu Lys Ser Leu Gln Leu Gln Val Gln Met Met

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Ser Met Gly Tyr Gly Met Val Pro Met Met Phe Pro Gly Ile Gln Gln

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Tyr Met Pro Pro Met Gly Met Gly Ile Gly Met Gly Met Gly Met Glu

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Claims

1. An application of a soybean bHLH transcription factor GmPIF1 gene in promoting synthesis of soybean isoflavone is disclosed, wherein a nucleotide sequence of the soybean bHLH transcription factor GmPIF1 gene is shown as SEQ ID No.1, and an amino acid sequence of a protein coded by the soybean bHLH transcription factor GmPIF1 gene is shown as SEQ ID No. 2.

2. The use of the soybean bHLH transcription factor GmPIF1 gene of claim 1 for promoting the synthesis of soybean isoflavones, wherein: the GmPIF1 gene promotes the accumulation of isoflavones in the soybean hairy root.

3. The use of the soybean bHLH transcription factor GmPIF1 gene of claim 2 for promoting the synthesis of soybean isoflavones, wherein: the transcription factor GmPIF1 gene is transferred into soybean hairy roots to regulate and control the expression of related enzyme genes in an isoflavone synthesis pathway.