Beautiful millettia root CsbHLH9 transcription factor, method for increasing calycosin content and glucoside content and application of millettia root CsbHLH9 transcription factor

Abstract

The invention relates to a CsbHLH9 transcription factor of beautiful millettia roots, a method for increasing the content of calycosin and glucoside and application of the CsbHLH9 transcription factor, and belongs to the field of plant genetic engineering. Aiming at the problem that medicinal development is limited due to low contents of calycosin and glucoside thereof in beautiful millettia roots, the invention adopts the technical scheme that a CsbHLH9 transcription factor with a nucleotide sequence as shown in SEQ ID NO. 1 is provided. The transcription factor can be used for increasing the content of calycosin or / and calycosin-7-glucoside in plants, an overexpression vector pH7WG2D-CsbHLH9 containing the sequence is constructed, agrobacterium rhizogenes are converted, soybean cotyledon explants are infected, transgenic hairy roots are obtained through screening and identification, and the content of target components in the soybean hairy roots is remarkably increased. The transcription factor is mainly used for enhancing isoflavone biosynthetic gene expression, producing high-value medicinal components and providing a new way for millettia specisoa champ resource development.

Description

Technical Field

[0001] This invention belongs to the field of plant bioengineering technology, specifically relating to a type of *Smilax glabra*. CsbHLH9 Transcription factors and their methods and applications in increasing the content of verbascosides and glycosides. Background Technology

[0002] Niu Dali is a plant belonging to the genus *Spatholobus* in the legume family (Fabaceae). Callerya speciosa The dried root of *Champ. ex Benth.* Schott is listed in authoritative literature such as the *Guangxi Traditional Chinese Medicine Standards* (1990 edition), *Guangxi Zhuang Medicine Quality Standards* (Volume 1), *Chinese Zhuang Medicine*, and *Chinese Yao Medicine*. It is a traditional and iconic Zhuang and Yao medicine from Guangxi, and an important dual-use resource for both medicinal and edible purposes in the Lingnan region. In December 2024, *Champ. ex Benth.* Schott was included in the *List of Traditional Chinese Medicine Varieties Used in Guangxi Local Specialty Foods (First Batch)*. It is sweet and neutral in nature, and enters the lung and kidney meridians, possessing the effects of tonifying deficiency and moistening the lungs, strengthening muscles and tendons, and promoting blood circulation. Chemical composition studies show that the main active substances in *Champ. ex Benth.* Schott include flavonoids, alkaloids, coumarins, and organic acids. Among them, the main flavonoid components are verbascoflavonoids and their glycosides (verascoflavonoid glycosides), gentianin, and sophorin. Verbena isoflavones have been proven to possess a variety of biological activities, including antioxidant, anti-inflammatory, antibacterial, and antitumor effects. Furthermore, verbena isoflavone glycosides can be metabolized into verbena isoflavones in vivo, further expanding their pharmacological scope. However, the natural content of verbena isoflavones and their glycosides in *Smilax glabra* root is low, severely limiting the in-depth development of its medicinal value.

[0003] To overcome the limitations of natural resources, hairy root genetic transformation technology has been widely applied in the production of secondary metabolites due to its unique advantages. This technology integrates the T-DNA of the Agrobacterium rhizogenes Ri plasmid into the plant genome, inducing the production of genetically stable hairy roots. These roots not only efficiently synthesize secondary metabolites but also provide an ideal platform for gene function research. In recent years, utilizing transcription factors to regulate plant secondary metabolic pathways has become a core strategy for increasing the yield of effective components.

[0004] The bHLH (basic Helix-Loop-Helix) transcription factor family, as one of the largest transcription factor classes in plants, has yet to have its regulatory mechanisms on the synthesis of isoflavones and their glycosides in *Smilax glabra*, especially... CsbHLH9 The role of transcription factors in this process. Therefore, identifying key bHLH transcription factors that specifically regulate the synthesis of verrucoside isoflavones and their glycosides, and establishing an efficient production system, is of great significance for enhancing the medicinal value of *Smilax glabra*. Summary of the Invention

[0005] To fill the technical gap in the regulation of calycosin and its glycosides biosynthesis by transcription factors and break through the resource limitation of the low natural content of target components in Millettia speciosa Champ., the present invention provides a molecular regulation method for specifically activating isoflavone synthesis genes by transcription factors ( CsbHLH9 ), applications, and achieving efficient accumulation of active substances, as well as recombinant vectors and transgenic soybean hairy roots involved in the application process.

[0006] To achieve the purpose of the present invention, a Millettia speciosa Champ. CsbHLH9 transcription factor has a nucleotide sequence as shown in SEQ ID NO.1.

[0007] Furthermore, the Millettia speciosa Champ. CsbHLH9 transcription factor encodes a protein with an amino acid sequence as shown in SEQ IDNO:2.

[0008] The present invention provides an application of a Millettia speciosa Champ. CsbHLH9 transcription factor in increasing the content of calycosin or / and its glycosides in plants, wherein CsbHLH9 the nucleotide sequence of the transcription factor is as shown in SEQ ID NO:1, and the amino acid sequence of the encoded protein is as shown in SEQ ID NO:2; calycosin is calycosin, and the glycoside is calycosin glycoside.

[0009] The present invention provides a method for increasing the content of calycosin or / and its glycosides in soybean hairy roots, comprising the following steps: a) Construct an overexpression vector of the Millettia speciosa Champ. CsbHLH9 transcription factor containing the nucleotide sequence shown in SEQ ID NO:1; b) Use the overexpression vector obtained in step a) to transform Agrobacterium rhizogenes C58C1; c) Infect soybean cotyledon explants with Agrobacterium rhizogenes containing the overexpression vector to induce transgenic hairy roots; d) Screen and identify positive hairy root lines with overexpression CsbHLH9 ; e) Culture the positive hairy root lines to obtain hairy roots with high contents of calycosin or / and calycosin glycoside.

[0010] Furthermore, the method: In step a), the overexpression vector is pH7WG2D- CsbHLH9 , and the CsbHLH9 gene is cloned into the pH7WG2D vector through Gateway technology for construction; In step c), the explant is a soybean cotyledon pre-cultured for 2 days, the infection solution contains 100 μM acetosyringone, and the co-culture condition is dark culture at 25°C for 3 days; In step d), the screening is carried out using a 1 / 2 MS medium containing 30 mg / L kanamycin, and the identification methods include PCR amplification and green fluorescence detection; among the primers used for PCR amplification Forward primer 35S-F: 5’-GACGCACAATCCCACTATCC-3’; Reverse primer CsbHLH9 -det-R: 5’-AACGTTGTCACCGGTTGGG-3’.

[0011] A recombinant vector provided by the present invention contains the CsbHLH9 gene coding sequence shown in SEQ ID NO:1, and the recombinant vector is an overexpression vector pH7WG2D- CsbHLH9 .

[0012] A transgenic soybean hairy root provided by the present invention is characterized in that it is obtained by the method described in any one of the above, overexpresses the CsbHLH9 gene and the contents of calycosin and calycosin glycoside are significantly higher than those of wild-type hairy roots.

[0013] Furthermore, in the transgenic soybean hairy roots described above, the expression levels of the key enzyme genes for isoflavone synthesis in the hairy roots GmCHI , GmIFS , GmIFR are significantly up-regulated.

[0014] The present invention at least includes the following beneficial effects: 1. By constructing an overexpression vector and a genetic transformation system for soybean hairy roots, the present invention first proves that the CsbHLH9 transcription factor derived from Millettia speciosa Champ., with the nucleotide sequence as SEQ ID NO.1 and the amino acid sequence as SEQ ID NO.2, has a significant positive regulatory effect on the biosynthesis of calycosin and calycosin glycoside.

[0015] 2. In the transgenic soybean hairy roots overexpressing CsbHLH9 , the content of calycosin is increased by 33 - 36 times, and the content of calycosin glycoside is increased by 17 - 19 times, breaking through the limitation of low content in natural plants and providing a new way for large-scale production.

[0016] 3. Overexpression of the present invention CsbHLH9 can extremely significantly up-regulate the expression of the key enzyme genes in the soybean isoflavone synthesis pathway GmCHI、 GmIFS、GmIFR , indicating its molecular mechanism of promoting the synthesis of target products by regulating downstream genes.

[0017] 4. Using the genetic transformation technology mediated by Agrobacterium rhizogenes, the present invention successfully constructs CsbHLH9The overexpression soybean hairy root system has an induction rate of nearly 73%, and has the advantages of genetic stability, short culture cycle and high secondary metabolite enrichment efficiency, and provides a technical basis for directly producing high-value flavonoid components.

[0018] 5, The application not only provides a key target for the synthetic biology research of medicinal components of Millettia pachycarpa Benth, but also lays a theoretical support for producing calycosin and its glycoside element by using the soybean hairy root bioreactor, and accelerates the innovation and industrial application of Millettia pachycarpa germplasm resources.

[0019] Other advantages, objects and features of the present application will be apparent from the following description, and will be understood by those skilled in the art through the study and practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 : RNA electrophoresis result diagram (A) and CsbHLH9 electrophoresis detection schematic diagram of gene PCR amplification (B); Figure 2 : vector pH7WG2D vector structure diagram; Figure 3 : pH7WG2D- CsbHLH9 recombinant plasmid bacterial liquid PCR electrophoresis diagram; Figure 4 : morphological diagram of soybean hairy root; Figure 5 : overexpression CsbHLH9 identification of root system, wherein M, marker;1-3 respectively represent the overexpression CsbHLH9 root system still surviving after screening culture; Figure 6 : green fluorescent detection of hairy root;WT, wild type soybean root (negative control);pH7WG2D, empty load hairy root;K7WG2D- CsbHLH9 , overexpression CsbHLH9 hairy root; Figure 7 : CsbHLH9 schematic diagram of expression analysis of gene, wherein, EV: empty load hairy root;OE4, OE8, OE19 respectively represent overexpression strains OE4, OE8, OE19;** represents P < 0.01; Figure 8 : expression analysis of key enzyme genes in soybean isoflavone biosynthesis path, wherein, EV: empty load hairy root;OE4, OE8, OE19 respectively represent overexpression strains OE4, OE8, OE19;** represents P < 0.01; Figure 9 : overexpression CsbHLH9The effects of verbascoside and verbascoside glycoside content were analyzed. EV: empty hairy root; OE4, OE8, and OE19 represent overexpression lines OE4, OE8, and OE19, respectively; ** indicates P < 0.01. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to examples, so that those skilled in the art can implement it based on the description.

[0022] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0023] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0024] Example 1: Construction of CsbHLH9 transcription factor overexpression vector (1) CsbHLH9 Gene Fragment Acquisition In the genome of *Smilax glabra* CsbHLH9 Specific primers were designed based on the CDS sequences of transcription factors. CsbHLH9 -F and CsbHLH9 -R was synthesized by Shanghai Sangon Biotech Co., Ltd.

[0025] The nucleotide sequences of the primers are shown below: CsbHLH9 -F:5'-ATGAAGGTTGAGGTGGGGT-3'.

[0026] CsbHLH9 -R:5'-CTACCCAACCGGTGACAAC-3'.

[0027] Electrophoretic analysis of total RNA from *Smilax glabra* root revealed that both the 18S and 28S bands were intact and clear, indicating that the RNA extracted in this invention was not degraded. Figure 1 A) This meets the experimental requirements. Using cDNA from *Smilax glabra* root as a template, the following steps were performed: CsbHLH9 Gene fragment amplification, target fragment amplification system: 2×PhantaMax Buffer, 25 μL; dNTPMix (10 mmol / L), 1 μL; CsbHLH9 -F (10 μmol / L), 2 μL; CsbHLH9R (10 μmol / L), 2 μL; Phanta Max Super-Fidelity DNA Polymerase, 1 μL; cDNA, 2 μL; ddH2O, 17 μL; total 50 μL. The amplification procedure was as follows: 95℃, 3 min; 94℃, 30 s, 58℃, 30 s, 72℃, 1 min, 32 cycles; 72℃, 10 min, 4℃ constant temperature preservation. After PCR amplification, 1% agarose gel electrophoresis was used to detect the PCR product, and the results were as shown in Fig. 1B. Figure 1 As shown in Fig. 1B, the size of the target fragment obtained by PCR amplification was about 1000-2000 bp. Figure 1 As shown in Fig. 1B, the size of the target fragment obtained by PCR amplification was about 1000-2000 bp. CsbHLH9 The complete open reading frame (ORF) of the transcription factor gene was 1758 bp, which encoded 586 amino acids. CsbHLH9 The nucleotide sequence of the transcription factor gene is shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2.

[0028] (2) Intermediate vector pDNOR221- CsbHLH9 Construction attB sites were added to both sides of the CDS sequence of pDNOR221- CsbHLH9 by two rounds of PCR reaction, so that attB sites were added to both ends of the fragment to form attB- CsbHLH9 recombination PCR product. PMD19-T- CsbHLH9 was used as a template for PCR amplification, and the PCR amplification system was as follows: 2×Phanta Max Buffer, 25 μL; dNTP Mix (10 mmol / L), 1 μL; CsbHLH9 -F (10 μmol / L), 2 μL; CsbHLH9 -R (10 μmol / L), 2 μL; Phanta Max Super-Fidelity DNA Polymerase, 1 μL; cDNA, 2 μL; ddH2O, 17 μL; total 50 μL. The PCR amplification procedure was as follows: 95℃, 3 min; 94℃, 30 s, 58℃, 30 s, 72℃, 1 min, 32 cycles; 72℃, 10 min, 4℃ constant temperature preservation.

[0029] The nucleotide sequences of the first round of PCR primers are as follows: CsbHLH9 -F: 5'-AAAAAGCAGGCTCCATGAAGGTTGAGGTGGGGT-3'.

[0030] CsbHLH9 - R: 5'-AGAAAGCTGGGTTCTACCCAACCGGTGACAAC-3'.

[0031] The nucleotide sequences of the second round of PCR primers are as follows: Adapter attB1: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3'.

[0032] Adapter attB2: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTT-3'.

[0033] The PCR product was subjected to agarose gel electrophoresis and the target band with a size of about 1200 bp was recovered. The BP recombination reaction system was prepared on ice: intermediate vector, 0.5 μL; target fragment, 1 μL; BP clonase, 0.5 μL; ddH2O, 0.5 μL; a total of 2.5 μL. After incubation at 25°C overnight, 1 μL of Proteinase K was added to the system and incubated at 37°C for 10 min to terminate the reaction; after the reaction was completed, it was cooled on ice, and the recombination product was obtained. The intermediate vector product was transformed into DH5α E. coli, and plated on LB solid medium containing Kan and incubated at 30°C overnight. The colonies were picked and subjected to PCR to verify their positivity, and the positive bacterial solution was sent to the company for sequencing. The sequencing results were successfully compared, the plasmid was extracted and preserved with 50% glycerol, and stored at -80°C. The primers used in the colony PCR detection are as follows: M13F: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'.

[0034] M13R: 5'-AGCGGATAACAATTTCACACAGGA-3'.

[0035] The sequencing results showed that the intermediate vector pDNOR221- CsbHLH9 was successfully constructed.

[0036] (3) Overexpression vector pH7WG2D- CsbHLH9 Recombination reaction The CDS sequence of CsbHLH9 was assembled into the pH7WG2D overexpression vector using the homologous recombination method of Gateway technology (vector map as shown in Figure 2The LR recombination reaction system was prepared on ice: 0.5 μL of the intermediate vector plasmid containing the target fragment; 0.5 μL of the overexpression vector pH7WG2D; 0.5 μL of LR clonase; 1.5 μL of ddH2O; and a total of 3 μL. After the reaction was carriedied out overnight on a floating plate in a 25°C water bath, 1 μL of Proteinase K was added to terminate the reaction at 37°C for 10 min. After the reaction was completed, the system was cooled on ice, and the recombination product was obtained.

[0037] (4) Transformation of the recombination product into E. coli The E. coli DH5α competent cells were thawed on ice, 3 μL of the recombination product was added, and the mixture was mixed by flicking. The mixture was subjected to ice bath for 30 min, 42°C heat shock for 1 min, and ice bath for 2 min. 500 μL of LB liquid medium was added, and the mixture was incubated at 37°C and 200 rpm for 45 min. The mixture was centrifuged at 12,000 rpm for 2 min, and 400 μL of supernatant was discarded. The bacterial cells were resuspended by gently blowing with a pipette. The bacterial cells were spread on LB solid medium (containing 50 mg / L Spe), and incubated at 37°C for 1 h, and then incubated for 12-16 h.

[0038] (5) Identification of the recombination product A single colony on the LB plate was picked up with a sterilized toothpick, and placed in 50 μL of ddH2O. 1 μL of the bacterial solution was used as a template for PCR amplification. A 20 μL system was used, and the system was as follows: 10 μL of 2×PCR Mix, 0.3 μL of primer-F, 0.3 μL of primer-R, 1 μL of template, and 8.4 μL of ddH2O.

[0039] The nucleotide sequences of the primers are as follows: 35S-F: 5'-GACGCACAATCCCACTATCC-3'.

[0040] CsbHLH9 -det-R: 5'-AACGTTGTCACCGGTTGGG-3'.

[0041] The PCR program was as follows: 94°C for 10 min→ (94°C for 30 s→ 55°C for 30 s→ 72°C for 30 s) for 32 cycles→ 72°C for 5 min→ 4°C for 10 min. The PCR product was detected by electrophoresis in a 1% agarose gel at 100 V for 30 min. Figure 3 The colonies that tested positive were sent to a gene company for sequencing. After successful sequencing, the bacterial solution was stored, and the recombination plasmid was extracted using a plasmid extraction kit, and the pH7WG2D- CsbHLH9 recombination plasmid was successfully obtained.

[0042] Example 2: Induction of soybean hairy roots The specific steps of genetic transformation of soybean hairy roots are as follows: (1) Preparation of Agrobacterium infection solution Agrobacterium-mediated transformation was used to transfer plasmids pH7WG2D and pH7WG2D- CsbHLH9 Transformed into *Agrobacterium rhizogenes* C58C1 competent cells. In a sterile environment, pick up the transformed *Agrobacterium rhizogenes* C58C1 cells using an inoculation loop and streak them onto LB solid medium containing 50 mg / L spectinomycin, incubating upside down at 28 °C for 48–72 h. Pick a single colony and inoculate it into 1.5 mL of LB liquid medium (containing 50 mg / L spectinomycin), incubating at 220 rpm and 28 °C for 16–18 h. Inoculate 0.8%–1.0% (v / v) into 50 mL of LB liquid medium (containing 50 mg / L spectinomycin), incubating at 28 °C and 220 rpm. When the bacterial concentration reaches OD500... 600 When the bacterial growth rate is approximately 0.8–1.0, centrifuge at 8000 rpm for 5 min, discard the supernatant; resuspend the bacteria in an equal volume of fresh MS liquid medium, add 100 μM acetylsuccinone, and incubate at room temperature for 1–2 h to obtain the infection solution.

[0043] (2) Preparation of explants Select soybean seeds with intact seed coats and sterilize them in 0.1% mercuric chloride solution for 15 min. After washing five times with sterile water, blot dry with filter paper and place in 1 / 2 MS medium. After soybean germination, make incisions in the cotyledons with a scalpel and pre-culture in 1 / 2 MS medium in the dark for 2 days.

[0044] (3) Agrobacterium infection Agrobacterium rhizogenes was placed in 50 mL of YEB culture medium and incubated overnight at 28°C and 200 rpm until OD reached. 600 = 0.6 ~ 0.8. Centrifuge Agrobacterium at 4000 rpm for 10 min and discard the supernatant. Add 50 mL of 1 / 2 MS + 100 μM AS culture medium, resuspend, and incubate at 100 rpm and 28℃ for 30 min. Add the scratched soybean cotyledons and incubate at 100 rpm and 28℃ for 30 min.

[0045] (4) Co-cultivation After infection, the bacterial solution on the leaf surface was dried with sterile filter paper, and then transferred to 1 / 2 MS solid medium containing 100 μM acetylsyl syringone and incubated in the dark at 25 ℃ for 3 days.

[0046] (5) Antibacterial culture During the co-cultivation, the plaque-forming explants are washed with sterile water containing 300-400 mg / L carbenicillin and 150-250 mg / L timentin for 2-3 times, the surface water is absorbed with sterile filter paper, and the explants are inoculated into 1 / 2 MS + 400 mg / L cef medium for antibacterial culture in the dark. The culture is subcultured every other week, and the concentration of the antibacterial antibiotic is gradually reduced until the plaque around the stem segments completely disappears. About 8-12 days later, white hairy roots about 2-3 cm long are grown, and the hairy roots are identified and transferred after 30 days of continuous culture.

[0047] (6) Screening and identification of positive hairy roots The hairy roots about 2-3 cm long are cut and transferred to 1 / 2 MS solid medium containing the screening antibiotic (30 mg / L kanamycin) for dark culture at 28°C. During the culture, transgenic hairy roots with kanamycin resistance, faster growth and more branches are screened Figure 4 . The screened hairy roots are transferred to 1 / 2 MS solid medium for expansion culture, and PCR identification analysis Figure 5 and green fluorescence detection analysis Figure 6 are performed after 30 days of culture. The hairy roots obtained by infection with Agrobacterium containing the overexpression vector are overexpression hairy roots (OE), and the hairy roots obtained by infection with Agrobacterium containing the empty vector are empty-type hairy roots (EV).

[0048] Example 3 Identification of overexpression CsbHLH9 hairy root positive strains The soybean hairy roots obtained in Example 2 are taken, and DNA is extracted. According to the 35S promoter contained in the vector, an upstream primer 35S-F is designed, according to the coding sequence of the CsbHLH9 gene, a downstream primer CsbHLH9 -det-R is designed, and the extracted DNA is used as a template for PCR detection.

[0049] The nucleotide sequences of the primers are as follows: 35S-F: 5'-GACGCACAATCCCACTATCC-3' (targeting the 35S promoter of the vector) CsbHLH9 -det-R: 5'-AACGTTGTCACCGGTTGGG-3' (targeting the coding region) CsbHLH9 The PCR amplification system is as follows: 2×PCR mix, 10 μL; upstream primer (10 μmol / L), 0.5 μL; downstream primer (10 μmol / L), 0.5 μL; DNA, 2 μL; and ddH2O is added to 20 μL. ​

[0050] The amplification procedure is as follows: 94°C, 10 min (pre-denaturation); 94°C, 30 s; 55°C, 30 s; 72°C, 30 s, 32 cycles; 72°C, 5 min, 4°C storage.

[0051] The results of electrophoretic detection of the PCR products are shown in Figure 5 Fig. 2. All three hairy root lines have bands, and the size of the PCR products is about 1758 bp, which is consistent with the size of the target band (ORF). The induction rate of each transgenic hairy root was found to be about 73% (Table 1). Three positive lines were randomly selected for further experiments, and the numbers of the overexpression lines were OE4, OE8, and OE19. CsbHLH9

[0052] Table 1 Transgenic hairy root induction rate Table 1 Transgenic hairy root induction rate Example 4 CsbHLH9 Expression analysis of key enzyme genes in the soybean isoflavone biosynthetic pathway qRT-PCR was used to detect CsbHLH9 transcription factor genes and key enzyme genes in the soybean isoflavone biosynthetic pathway GmCHI , GmIFS , GmIFR The expression levels in the overexpression hairy roots (overexpression lines OE4, OE8, and OE19) and the empty vector hairy roots (EV) were determined using GAPDH as the internal reference gene. The primers used in the qRT-PCR detection are shown in Table 2.

[0053] Table 2 Validation of qPCR primers for hairy roots Table 2 Validation of qPCR primers for hairy roots The reaction system is as follows: SYBR Green qPCR Mix (2x), 5 μL; forward primer (10 μmol / L), 0.3 μL; reverse primer (10 μmol / L), 0.3 μL; cDNA template, 2 μL; RNase-free ddH2O, 2.4 μL; and a total of 10 μL.

[0054] ​The qRT-PCR reaction conditions are as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 15 s, annealing at 60℃ for 30 s, extension at 72℃ for 1 min, 34 cycles.

[0055] The results are shown in Table 1. Figure 7 Compared with the EV (empty vector), the expression levels of the genes and the key enzyme genes of the soybean isoflavone biosynthetic pathway Figure 8 in the overexpression strains OE4, OE8 and OE19 were significantly increased. CsbHLH9 The fold difference was 5.2-5.8, reaching a very significant level (**P < 0.01); the overexpression of GmCHI , GmIFS , GmIFR the hairy roots can up-regulate the expression of the isoflavone biosynthetic genes of soybean CsbHLH9 , GmIFR , GmIFS , GmCHI and the like (**P < 0.01). It can be seen that CsbHLH9 the overexpression of the genes can significantly activate the expression of the key enzyme genes of the soybean isoflavone biosynthetic pathway.

[0056] Example 5: Determination of the contents of calycosin and calycosin glycoside (1) Preparation of the control solution and preparation of the standard curve Accurately weigh 1.02 mg of calycosin (analytical purity, purity ≥98) and 1.02 mg of calycosin glycoside (analytical purity, purity ≥98) into a 10 mL volumetric flask, and add 80% methanol aqueous solution to the calibration mark of the volumetric flask to obtain the stock solution of the control solution. Filter the stock solution through a 0.22 μm organic filter membrane, seal with a sealing film, and store at 4℃. Take 1.5 mL of each of the two stock solutions and add them together into a 10 mL volumetric flask, and add 80% methanol aqueous solution to the calibration mark to obtain a mixed control solution of 15 µg / mL calycosin glycoside and 15 µg / mL calycosin. Dilute to 10, 20, 40, 60, 80, 100 and 150 times to obtain 1#-7# mixed control solutions. Sample injection according to the chromatographic condition method, and calculate the standard curve function using the external standard method, which is then used for component content calculation. Draw the standard curve with the concentration of the control (µg / mL) as the abscissa x and the peak area as the ordinate y. The standard curves are as follows: calycosin: y = 31807.5x + 163.595, R 2 = 0.9999; calycosin glycoside: y = 20373.1x + 164.855, R 2 = 0.9999.

[0057] (2) Extraction of calycosin and calycosin glycoside from soybean hairy roots The OE-4, 8, 19 and EV transgenic hairy roots were dried at 45 ℃, ground into powder by liquid nitrogen drying freezing method, and each group of transgenic hairy root powder was weighed accurately in triplicate. 700 μL of 80% methanol solution was used to ultrasonically extract 3 mg of transgenic powder (power 300 W, frequency 40 kHz) for 1 h. After ultrasonic extraction, centrifugation (12 000 rpm, 10 min) was performed, and the supernatant was filtered through a 0.22 μm organic filter membrane into a sample injection bottle, and the solution was the test sample solution.

[0058] (3) Content analysis of calycosin and calycosin glycoside Agela Venusic C18 (250 mm×4.6 mm, 5 μm) was used as the chromatographic column; (A) acetonitrile-(B) 0.1% phosphoric acid solution was used as the mobile phase for gradient elution (gradient 1: 0-30 min, 10%-35% A; 30-40 min, 35%-55% A; 40-50 min, 55%-70% A; 50-55 min, 70%-10% A), the flow rate was 1.0 mL / min, the column temperature was 30 ℃, the wavelength was 205 nm, and the column temperature was 30 ℃. The injection amount of the test sample solution was 10 μL, and the injection amount of the standard curve preparation was divided into 2.5, 5, 7.5, 10, 12.5 μL. The sample solution injection peak area was determined, and the content was calculated according to the standard curve.

[0059] The experimental results are shown in Table 1. Figure 9 Compared with EV (empty load), the contents of calycosin and calycosin glycoside in the overexpression strains OE4, 8 and 19 were increased by 33-36 times and 17-19 times respectively (**P<0.01).

[0060] The above examples show that the transcription factor gene CsbHLH9 is cloned from the leaves of Millettia pachyloba, and the overexpression CsbHLH9 vector is constructed. The transgenic soybean hairy root system overexpressing CsbHLH9 is obtained by using the genetic transformation method mediated by Agrobacterium rhizogenes (C58C1). The experimental results show that, compared with the control root system, the expression levels of the key enzyme genes CsbHLH9 , GmIFR , GmIFS , GmCHI of the isoflavone biosynthesis pathway in the soybean hairy roots overexpressing CsbHLH9 are significantly activated. At the same time, the contents of calycosin and calycosin glycoside in the roots overexpressing CsbHLH9 are significantly increased.

[0061] SEQ ID NO. 1 SEQ ID NO. 2 MKVEVGSGRVIWDDEEKGMVAAVLGARALEFLMTKSVSNESVLMAVGSDDGLQNKLSDLVDRPNGSNFSWNYSIFWQLSQSKSGEWVLGWGDGCCREPNEEEEGGLSLRIEDGMQQSMRKRVLQKLHTAFGGSDEDNYAFGLDRVTDTEMFFLASMYFSFPRGHGGPGKCFASGKHLWLKSVSDYCVRSFLAKSAGIQTVVLVPTDLGVVELGSVRMLPESFELLHAVKSVFSTQASVNDERDEGAIGSSKVGGVVNTGRAHFREKLAIRKMEDRPWGGLPNGNNNNNSNNSINFLHARNGFHGSSWGGVNQGVGLRQPGPAEIFAPRSSAGHVPEVANGARQDFRLNNNYEQPPQRQVQMQIDFSGATSRPSVRPVIAESELPDCKEDQPSAADERRPRKRGRKPANGREEPLNHVEAERQRREKLNQRFYALRSVVPNISRMDKASLLGDAIAYINELQAKLRVMESERERFGSTSRDGSVLEANSGSENHQNRAPDVDIEASQDEVIVRVSCPLDTHPVSKVIQTFKEAQISVVVSKLAAANDTVFHTFVIKSQGSEQLTKDKLIAAFSRESSSLQTLSPVG While embodiments of the application have been disclosed in connection with the specified embodiments, it should be understood that it can be adapted in a variety of arrangements. Still yet, various aspects and embodiments thereof can include combinations of the aspects and embodiments in other solutions. Other modifications of the application will occur to those skilled in the art upon reading the description. Therefore, it is to be understood that there is no intention to limit the application to the specific illustrative embodiments, but rather the intention is to cover all modifications of the application as would be within the skills and knowledge of those in the art.

Claims

1. A Withania somnifera CsbHLH9 Transcription factor characterized in that, The nucleotide sequence is shown as SEQ ID NO.

1.

2. The cowslip of claim 1 CsbHLH9 a transcription factor characterized in that, The amino acid sequence of the encoded protein is shown as SEQ ID NO:

2.

3. A cowhage CsbHLH9 The application of a transcription factor in increasing the content of pterostilbene or / and glycoside in a plant, wherein CsbHLH9 The nucleotide sequence of the transcription factor is shown as SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO: 2; the pterostilbene is pterostilbene, and the glycoside is pterostilbene glycoside.

4. A method for increasing the content of puerarin or / and glycosides in soybean hairy roots, characterized in that, The method comprises the following steps: a) constructing a bovine daidzein comprising the nucleotide sequence set forth in SEQ ID NO: 1 CsbHLH9 transcription factor overexpression vectors; b) transforming Agrobacterium rhizogenes C58C1 with the overexpression vector obtained in step a); c) infecting soybean cotyledon explants with Agrobacterium rhizogenes containing the overexpression vector to induce transgenic hairy roots; d) screening and identifying positive hairy root lines overexpressing CsbHLH9 the gene of interest; e) culturing positive hairy root lines to obtain hairy roots with high content of calycosin or / and calycosin glycosides.

5. The method of claim 2, wherein, The method comprises: In step a), the overexpression vector is pH7WG2D CsbHLH9 constructed by cloning the gene into the pH7WG2D vector using the Gateway technology. CsbHLH9 constructed by cloning the gene into the pH7WG2D vector using the Gateway technology. In step c), the explants are soybean cotyledons pre-cultured for 2 days, the infection solution contains 100 μM acetosyringone, and the co-culture condition is 25°C dark culture for 3 days; In step d), the screening uses 1 / 2 MS medium containing 30 mg / L kanamycin, and the identification method comprises PCR amplification and green fluorescence detection; the primers used for PCR amplification are upstream primer 35S-F: 5'-GACGCACAATCCCACTATCC-3'; Downstream primer CsbHLH9 - det-R: 5'- AACGTTGTCACCGGTTGGG -3'.

6. A recombinant vector, characterized in that, comprises a gene coding sequence shown as SEQ ID NO: 1, and the recombinant vector is overexpression vector pH7WG2D- CsbHLH9 comprises a gene coding sequence shown as SEQ ID NO: 1, and the recombinant vector is overexpression vector pH7WG2D- CsbHLH9 .

7. A transgenic soybean hairy root, characterized in that, obtained from the method of any one of claims 3-5, which overexpresses CsbHLH9 Genes and the content of calycosin and calycosin glycosides were significantly higher than those in wild-type hairy roots.

8. The transgenic soybean hairy roots of claim 6, wherein, Key enzyme genes for isoflavone synthesis in hairy roots GmCHI , GmIFS , GmIFR expression was significantly up-regulated.

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