Gynostemma pentaphylla transcription factor GpMYC65 and application thereof

Abstract

The invention discloses a gynostemma pentaphyllum transcription factor GpMYC65 and application thereof. The nucleotide sequence of the gynostemma pentaphyllum transcription factor GpMYC65 is as shown in SEQ ID NO. 1. The invention also discloses a preparation method of the gynostemma pentaphyllum transcription factor GpMYC65. The amino acid sequence of the protein encoded by the gynostemma pentaphylla transcription factor GpMYC65 is as shown in SEQ ID NO. 2. The heavy recombinant vector contains a gynostemma pentaphylla transcription factor GpMYC65. The invention relates to application of a Gynostemma pentaphyllum transcription factor GpMYC65 in regulating and controlling the content of dammarane type triterpenoid saponin in Gynostemma pentaphyllum. The application comprises the following steps: step 1, constructing an overexpression vector containing a transcription factor GpMYC65 encoding gene; 2, transforming the overexpression vector into gynostemma pentaphyllum leaves by using an agrobacterium-mediated method to obtain genetic transformation hairy roots, and screening and identifying positive hairy roots of an overexpression transcription factor GpMYC65; and 3, carrying out liquid culture on the screened positive hairy roots to obtain transgenic hairy roots of which the dammarane type triterpenoid saponin content is higher than that of the wild hairy roots. The content of dammarane type triterpenoid saponin can be remarkably increased by overexpressing the transcription factor GpMYC65 in the fiveleaf gynostemma herb hairy roots.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology. More specifically, the present invention relates to a Gynostemma pentaphyllum transcription factor GpMYC65 and its applications. Background Art

[0002] Gynostemma pentaphyllum Gynostemma pentaphyllum (Thunb.) Makino is an important characteristic medicinal plant resource in China. Gypenosides are the main active ingredients of Gynostemma pentaphyllum. Its chemical structure is dammarane-type tetracyclic triterpenoids, which have a wide range of pharmacological effects: 1. It can promote sleep and relieve anxiety; 2. It can lower blood sugar and blood lipids; 3. It also plays an important role in the treatment and prevention of cancer, diabetes and other chronic diseases. Gynostemma pentaphyllum is the only plant known to contain dammarane-type tetracyclic triterpenoid components other than the genus Panax in the Araliaceae family. Therefore, Gynostemma pentaphyllum is a good alternative resource for the production and acquisition of such saponins, and thus has great development and utilization value.

[0003] Currently, there are significant differences in the contents of the main active ingredients among different strains or ecotypes of Gynostemma pentaphyllum, and the phenomena of passing off inferior goods as good ones often occur; moreover, the breeding work of excellent germplasms of Gynostemma pentaphyllum with high contents of active ingredients has progressed slowly, seriously affecting the healthy and sustainable development of the Gynostemma pentaphyllum industry. Therefore, carrying out research on the molecular mechanism of the biosynthesis regulation of dammarane-type triterpenoid saponins in Gynostemma pentaphyllum, and excavating and identifying functional genes that regulate the biosynthesis of dammarane-type triterpenoid saponins in Gynostemma pentaphyllum can provide gene resources for improving the content of dammarane-type triterpenoid saponins in Gynostemma pentaphyllum by using molecular biology means, and provide a theoretical basis for the application of molecular breeding technology in the breeding of new varieties with high saponin content.

[0004] The main active ingredients of Gynostemma pentaphyllum are dammarane-type triterpenoid saponins, and their synthesis and accumulation are affected by various external environmental conditions. Environmental signals ultimately affect the accumulation of secondary metabolites by binding of transcription factors to cis-acting elements in the promoter regions of downstream structural genes to activate or inhibit the expression of related genes. Regulating the expression of key structural genes in the plant secondary metabolic pathway through transcription factors is an effective way to achieve the efficient synthesis and directional accumulation of effective components of traditional Chinese medicine. The present invention provides a Gynostemma pentaphyllum transcription factor GpMYC65. This transcription factor is a MYC-like regulator carrying a basic helix-loop-helix (bHLH) domain and plays an important role in the response and adaptation of plants to environmental changes. There has been no research report on the regulation of the biosynthesis of dammarane-type triterpenoid saponins in Gynostemma pentaphyllum by MYC-like transcription factors. Summary of the Invention

[0005] Another object of the present invention is to provide a Gynostemma pentaphyllum transcription factor GpMYC65.

[0006] To achieve these objects and other advantages according to the present invention, a Gynostemma pentaphyllum transcription factor GpMYC65 is provided, and the nucleotide sequence of the Gynostemma pentaphyllum transcription factor GpMYC65 is shown in SEQ ID NO.1.

[0007] The present invention also provides an amino acid sequence of a protein encoded by the Gynostemma pentaphyllum transcription factor GpMYC65, and the amino acid sequence of the protein encoded by the Gynostemma pentaphyllum transcription factor GpMYC65 is shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant expression vector, and the recombinant vector contains the Gynostemma pentaphyllum transcription factor GpMYC65.

[0009] The present invention also provides an application of the Gynostemma pentaphyllum transcription factor GpMYC65 in regulating the content of dammarane-type triterpenoid saponins in Gynostemma pentaphyllum.

[0010] Preferably, the dammarane-type triterpenoid saponins are selected from at least one of gypenoside XVII, gypenoside VN2, gypenoside XXIII or gypenoside Rd.

[0011] Preferably, it includes the following steps: Step 1: Construct an overexpression vector containing the coding gene of the transcription factor GpMYC65; Step 2: Use the Agrobacterium-mediated method to transform the overexpression vector into Gynostemma pentaphyllum leaves to obtain genetically transformed hairy roots, and screen and identify positive hairy roots overexpressing the transcription factor GpMYC65; Step 3: Perform liquid culture on the screened positive hairy roots to obtain transgenic hairy roots with a dammarane-type triterpenoid saponin content higher than that of wild-type hairy roots.

[0012] Preferably, the construction method of the overexpression vector in Step 1 includes the following steps: A1: Using Gynostemma pentaphyllum leaf cDNA as a template and GpMYC65-F and GpMYC65-R as primers, obtain the GpMYC65 gene fragment by PCR amplification; A2: Connect the GpMYC65 gene fragment to the intermediate vector pDNOR221 and recombine it into the overexpression vector pK2GW7 to obtain an overexpression vector containing the coding gene of the transcription factor GpMYC65.

[0013] The present invention has at least the following beneficial effects: The present invention creatively discovers that overexpressing the transcription factor GpMYC65 in the hairy roots of Gynostemma pentaphyllum can significantly increase the content of dammarane-type triterpenoid saponins. Furthermore, the present invention constructs an overexpression vector of the transcription factor GpMYC65 and uses the Gynostemma pentaphyllum hairy root system to verify its function of regulating dammarane-type triterpenoid saponins in Gynostemma pentaphyllum. Experiments show that the method of the present invention can effectively enhance the expression level of the transcription factor GpMYC65 in the hairy roots of Gynostemma pentaphyllum and positively regulate the expression of 4 key enzyme genes in the biosynthetic pathway of dammarane-type triterpenoid saponins. Among them, the increase in the expression level of the GpOSC1 gene is the most obvious, thus significantly promoting the biosynthesis and accumulation of dammarane-type triterpenoid saponins in the hairy roots of Gynostemma pentaphyllum. The present invention has important theoretical value for cultivating new germplasms of Gynostemma pentaphyllum with high content of active ingredients and has important industrial significance for promoting the sustainable development of the Gynostemma pentaphyllum medicinal material industry.

[0014] Other advantages, objectives, and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. Brief Description of the Drawings

[0015] Figure 1 Schematic diagram of the electrophoresis detection after PCR amplification of the GpMYC65 gene; Figure 2 Schematic diagram of the structure of the pK2GW7 overexpression vector; Figure 3 Electrophoresis diagram of the colony PCR of the pK2GW7-GpMYC65 recombinant plasmid; Figure 4 Schematic diagram of the process of transforming the overexpression vector (containing the coding gene of the transcription factor GpMYC65) into the leaves of Gynostemma pentaphyllum. Among them, A, the leaves of Gynostemma pentaphyllum are sterilized as explants and pre-cultured; B, hairy roots 2-3 cm long grow after 15 days of induction culture; C, hairy roots after 40 days of subculture; Figure 5 Identification of the rolB gene of the hairy root system. M, Markers; 1-12 respectively represent the roots overexpressing GpMYC65 that survived after screening culture; Figure 6 Identification of the rolC gene of the hairy root system. M, Markers; 1-12 respectively represent the roots overexpressing GpMYC65 that survived after screening culture; Figure 7 Identification of the roots overexpressing GpMYC65. M, Markers; 1-12 respectively represent the roots overexpressing GpMYC65 that survived after screening culture; Figure 8Schematic diagram of the expression analysis of key enzyme genes in the biosynthetic pathway of dammarane-type triterpenoid saponins. A is the schematic diagram of the expression analysis of the GpMYC65 gene, B is the schematic diagram of the expression analysis of the key enzyme gene GpFPS1, C is the schematic diagram of the expression analysis of the key enzyme gene GpSS1, D is the schematic diagram of the expression analysis of the key enzyme gene GpSE2, E is the schematic diagram of the expression analysis of the key enzyme gene GpSE3, F is the schematic diagram of the expression analysis of the key enzyme gene GpOSC1. Among them, EV represents the empty vector-type hairy roots; OE1, OE2, and OE3 respectively represent the overexpression lines OE1, OE2, and OE3, and ** represents P < 0.01; Figure 9 It is a multi-peak diagram for the detection of MRM metabolites in the negative ion mode; Figure 10 Among them, A is the phenotype diagram of the hairy roots of Gynostemma pentaphyllum overexpressing GpMYC65, B is the heat map of the relative contents of gypenoside XVII, gypenoside VN2, gypenoside XXIII, and gypenoside Rd in the hairy roots of Gynostemma pentaphyllum overexpressing GpMYC65, C is the change situation of the relative contents of gypenoside XVII, gypenoside VN2, gypenoside XXIII, and gypenoside Rd in the hairy roots of Gynostemma pentaphyllum overexpressing GpMYC65. Among them, EV: empty vector-type hairy roots; OE1, OE2, and OE3 respectively represent the overexpression lines OE1, OE2, and OE3; ** represents P < 0.01. Detailed implementation methods

[0016] The following further elaborates on the present invention in conjunction with the embodiments, so that those skilled in the art can implement it with reference to the text of the specification.

[0017] <Example 1> Construction of an overexpression vector containing the coding gene of the transcription factor GpMYC65: (1) Obtaining the GpMYC65 gene fragment Specific primers GpMYC65-F and GpMYC65-R were designed with reference to the CDS sequence of the Gynostemma pentaphyllum transcription factor GpMYC65 and synthesized by Shanghai Sangon Biotech Co., Ltd.

[0018] The nucleotide sequences of the specific primers are as follows: GpMYC65-F (SEQ ID NO.3): 5’-ATGAGTTATACATTTATTGTTATTTGCT-3’; GpMYC65-R (SEQ ID NO.4): 5’-TCAGCTTTCATTGATGAAACTCA-3’.

[0019] Using the cDNA of Gynostemma pentaphyllum leaves as a template, the GpMYC65 gene fragment was amplified. The amplification system for the target fragment was as follows: 2×Phanta Max Buffer, 25 μL; dNTP Mix (10 mmol / L), 1 μL; GpMYC65-F (10 μmol / L), 2 μL; GpMYC65-R (10 μmol / L), 2 μL; Phanta Max Super-Fidelity DNA Polymerase, 1 μL; cDNA, 2 μL; ddH2O, 17 μL; a total of 50 μL. The amplification program was: 95°C, 3 min; 94°C, 30 s, 60°C, 30 s, 72°C, 1 min, for 32 cycles; 72°C, 10 min, and stored at 4°C. After PCR amplification, the PCR products were electrophoretically detected using 1% agarose gel. The electrophoresis detection results are as Figure 1 shown. As Figure 1 can be seen, the size of the target fragment obtained by PCR amplification was shown to be between 1200 and 1300 bp, which was consistent with the size of the GpMYC65 gene. The target fragment was recovered using the Omega Gel Extraction Kit (D2500).

[0020] (2)Construction of the intermediate vector pDNOR221-GpMYC65 The attB sites were added to both sides of the CDS sequence of GpMYC65 through two rounds of PCR reactions to form an attB-GpMYC65 recombinant PCR product with attB sites at both ends of the fragment. The PCR amplification system: 2×Phanta Max Buffer, 25 μL; dNTP Mix (10 mmol / L), 1 μL; GpMYC65-F (10 μmol / L), 2 μL; GpMYC65-R (10 μmol / L), 2 μL; Phanta Max Super-Fidelity DNA Polymerase, 1 μL; cDNA, 2 μL; ddH2O, 17 μL; a total of 50 μL. The PCR amplification program was: 95°C, 3 min; 94°C, 30 s, 60°C, 30 s, 72°C, 1 min, for 32 cycles; 72°C, 10 min, and stored at 4°C.

[0021] The nucleotide sequences of the primers for the first round of PCR were as follows: GpMYC65-OX-F (SEQ ID NO.5): 5’-AAAAAGCAGGCTCCATGAGTTATACATTTATTGTTATTTGCT -3’; GpMYC65-OX-R (SEQ ID NO.6): 5’-AGAAAGCTGGGTTTCAGCTTTCATTGATGAAACTCA -3’.

[0022] The nucleotide sequences of the second-round PCR primers are as follows: Adapter attB1 (SEQ ID NO.7): 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3’; Adapter attB2 (SEQ ID NO.8): 5’-GGGGACCACTTTGTACAAGAAAGCTGGGT-3’.

[0023] The PCR products were subjected to agarose gel electrophoresis and the target band of about 1300 bp was recovered. A BP recombination reaction system was prepared on ice: intermediate vector, 0.6 μL; target fragment, 1 μL; BP clonase, 0.4 μL; ddH2O, 1 μL; total 3 μL. The reaction solution was gently pipetted and mixed, and briefly centrifuged to collect the reaction solution at the bottom of the tube, and reacted at 25 °C for 4 - 16 h; after the reaction was completed, it was cooled on ice to obtain the recombinant product. After the ligation was completed, Escherichia coli DH5α transformation, colony PCR, plasmid extraction and sequencing verification were carried out in sequence. The primers used for colony PCR are as follows: M13F (SEQ ID NO.9): 5’-CGCCAGGGTTTTCCCAGTCACGAC-3’; M13R (SEQ ID NO.10): 5’-AGCGGATAACAATTTCACACAGGA-3’.

[0024] The sequencing results showed that the intermediate vector pDNOR221-GpMYC65 was successfully constructed.

[0025] (3) Recombination reaction of overexpression vector pK2GW7-GpMYC65 The CDS sequence of GpMYC65 was assembled onto the pK2GW7 overexpression vector by homologous recombination using the Gateway technology (the vector map is as Figure 2 shown). An LR recombination reaction system was prepared on ice: intermediate vector pDNOR221-GpMYC65, 1.0 μL; overexpression vector, 0.6 μL; LR clonase, 0.4 μL; ddH2O, 1 μL; total 3 μL. The reaction solution was gently pipetted and mixed, and briefly centrifuged to collect the reaction solution at the bottom of the tube, and reacted at 25 °C for 4 - 16 h; after the reaction was completed, it was cooled on ice to obtain the recombinant product.

[0026] (4) Transformation of the recombinant product into Escherichia coli Thaw the competent Escherichia coli DH5α cells on ice, add 3 μL of the above-mentioned recombinant product, flick gently to mix evenly, and incubate on ice for 30 min. Heat shock at 42 °C for 1 min, then place on ice for 2 min. Add 500 μL of LB liquid medium, incubate at 37 °C with shaking at 200 rpm for 45 min. Then centrifuge at 12000 rpm for 2 min, discard 400 μL of the supernatant, and gently resuspend the bacteria with a pipette. Spread all the bacterial liquid on an LB solid medium (containing 50 mg / L Spe), incubate upright at 37 °C for 1 h, and then incubate upside down for 12 - 16 h.

[0027] (5) Identification of the recombinant product Pick a monoclonal colony on the LB plate with a sterilized toothpick, place it in 50 μL of ddH2O, and take 1 μL of this bacterial liquid as a template for PCR amplification. Use a 20 μL system as follows: 2×PCR Mix 10 μL, primer-F 0.3 μL, primer-R 0.3 μL, Template 1 μL, ddH2O 8.4 μL.

[0028] The nucleotide sequences of the primers are as follows: 35S-F (SEQ ID NO.11): 5’-GACGCACAATCCCACTATCC-3’; GpMYC65-det-R (SEQ ID NO.12): 5 '-TCAGCTTTCATTGATGAAACTCA-3’.

[0029] The PCR program is: 94 °C, 10 min → (94 °C, 30 s → 55 °C, 30 s → 72 °C, 30 s) 32 cycles → 72 °C, 5 min → 4 °C, 10 min. The PCR products are detected by electrophoresis in a 1% agarose gel at 100 V for 30 min (the electrophoresis pattern is as Figure 3 shown). The colonies verified as positive are sent to the gene company for sequencing. After successful sequencing, the bacterial liquid is preserved, and the recombinant plasmid is extracted using a plasmid extraction kit to successfully obtain the pK2GW7-GpMYC65 recombinant plasmid (i.e., the overexpression vector containing the coding gene of the transcription factor GpMYC65, which is also the recombinant expression vector).

[0030] <Example 2> Obtaining and screening of genetically transformed hairy roots. The genetic transformation steps of Gynostemma pentaphyllum are as follows, and the process schematic diagram is as Figure 4 shown: (1) Preparation of the Agrobacterium infection solution Select K599 as the infecting strain, and transform the constructed recombinant plasmid pK2GW7-GpMYC65 into Agrobacterium rhizogenes K599 by electroporation; in the sterile operating table, use an inoculation loop to pick up the transformed Agrobacterium rhizogenes K599 and streak it onto an LB solid medium containing 50 mg / L spectinomycin, and incubate it at 28 °C in an inverted position for 48 - 72 h; pick a single colony and inoculate it into 1.5 mL of LB liquid medium (containing 50 mg / L spectinomycin), culture it at 220 r / min and 28 °C for 16 - 18 h; inoculate it into 50 mL of LB liquid medium (containing 50 mg / L spectinomycin) according to an inoculation amount of 0.8% - 1.0%, at 28 °C and 220 r / min; when the bacterial liquid concentration reaches OD 600 is about 0.8 - 1.0, centrifuge at 8000 rpm for 5 min, pour off the supernatant; resuspend the bacteria with an equal volume of fresh MS liquid medium, add 100 μM acetosyringone, and place it at room temperature for 1 - 2 h to obtain the infection solution.

[0031] (2) Preparation of explants Select healthy Gynostemma pentaphyllum leaves, first disinfect them with 70% alcohol for 20 - 30 s, then soak them in 3% sodium hypochlorite solution for 10 - 15 min, and finally rinse them with sterile water 3 - 5 times; dry the leaf moisture with sterile filter paper, cut the leaves into small pieces of 0.8 - 1.0 cm × 0.8 - 1.0 cm, and place them in an MS solid medium containing 100 μM acetosyringone for dark culture for 48 h, and the culture temperature is 25 °C (as Figure 4 shown in A).

[0032] (3) Agrobacterium infection Transfer the pre-cultured Gynostemma pentaphyllum leaves to the prepared infection solution, place them on a shaker, shake at 90 r / min and 28 °C for 10 - 30 min to ensure sufficient contact between the pre-cultured leaves and Agrobacterium rhizogenes.

[0033] (4) Co-culture After the infection is completed, dry the bacterial liquid on the leaf surface with sterile filter paper, and then transfer it to an MS solid medium containing 100 μM acetosyringone for dark culture at 25 °C for 2 - 3 d.

[0034] (5) Washing bacteria Wash the explants that form bacterial plaques during co-culture 2 - 3 times with sterile water (containing 300 - 400 mg / L carbenicillin and 150 - 250 mg / L timentin), and then dry the moisture on the stem segment surface with sterile filter paper.

[0035] (6) Induction of hairy roots Transfer the explants to 6,7-V solid medium (containing 300 mg / L carbenicillin, 200 mg / L ticarcillin and 100 μM acetosyringone), and culture them at 28°C under long-day conditions (16 h light / 10 h dark). Subculture once every week, and gradually reduce the concentration of the antibacterial antibiotics until the bacterial plaque around the stem segments completely disappears. Hairy roots will start to grow after about 8 - 12 days( Figure 4 B), that is, genetically transformed hairy roots are obtained.

[0036] (7) Screening of positive hairy roots When the induced hairy roots grow to about 2 - 3 cm, cut off the hairy roots and transfer them to 6,7-V solid medium containing the screening antibiotic (30 mg / L kanamycin), and culture them in the dark at 28°C. During the culture process, transgenic hairy root systems with fast growth rate and many branches and kanamycin resistance are screened out. Transfer the screened hairy roots to solid 6,7-V medium for scale-up culture, and perform subsequent analysis after about 40 days of culture (such as Figure 4 shown in C). Among them, the hairy roots obtained by infecting with the Agrobacterium tumefaciens liquid containing the overexpression vector are overexpressed hairy roots (OE), and the hairy roots obtained by infecting with the Agrobacterium tumefaciens liquid containing the empty vector are empty vector hairy roots (EV).

[0037] <Example 3> Identification of positive hairy roots overexpressing the transcription factor GpMYC65: Take the Gynostemma pentaphyllum hairy roots (OE) cultured in Example 2 and extract DNA. Identify the Gynostemma pentaphyllum hairy roots according to the rolB and rolC genes on the pRi2659 plasmid in Agrobacterium rhizogenes K599. Design primers between the 35S promoter and the target gene GpMYC65, and use the extracted DNA as a template to detect the target gene by PCR.

[0038] The nucleotide sequences of the primers are as follows: rolB-F (SEQ ID NO.13): 5’-AGTCGCCGAGGTTTCTTTCTT-3’; rolB-R (SEQ ID NO.14): 5’-GCGAGAAGATGCAGAAAGTACTG-3’; rolC-F (SEQ ID NO.15): 5’-GCGGAATTTGACCTATGTGCT-3’; rolC-R (SEQ ID NO.16): 5’-ATTTGCATTCGCCATGCC-3’; 35S-F (SEQ ID NO.11): 5’-GACGCACAATCCCACTATCC-3’; GpMYC65-det-R (SEQ ID NO.12): 5’-TCAGCTTTCATTGATGAAACTCA-3’.

[0039] The PCR amplification system was as follows: 2×PCR mix, 10 μL; forward primer (10 μmol / L) of all target genes in this example, 0.5 μL; reverse primer (10 μmol / L), 0.5 μL; DNA, 2 μL; supplemented with ddH2O to 20 μL.

[0040] The amplification program was: 95°C, 3 min; 94°C, 30 s, 55°C, 30 s, 72°C, 1 min, for 32 cycles; 72°C, 10 min, stored at 4°C.

[0041] The PCR products were detected by electrophoresis, and the primers rolB-F / R and rolC-F / R were used to determine whether the hairy roots were Gynostemma pentaphyllum hairy roots induced by Agrobacterium rhizogenes K599. As Figure 5 shown, bands with a size of about 450 bp were detected in all 12 regenerated plant lines, which was consistent with the designed size of the rolB target band; in addition, as Figure 6 shown, bands with a size consistent with the designed rolC target band (about 550 bp) were detected in all 12 regenerated plant lines. The primers 35S-F and GpMYC65-det-R were used to determine the GpMYC65 overexpressing positive hairy roots. The results were as Figure 7 shown. A total of 12 pK2GW7-GpMYC65 single root systems were isolated, among which 10 were positive lines, and the positive rate was 83.3%. Three positive lines were randomly selected for the next experiment and re-numbered as overexpressing lines OE1, OE2, and OE3.

[0042] <Example 4> Expression analysis of key enzyme genes in the biosynthetic pathway of dammarane-type triterpenoid saponins: The expression levels of the transcription factor GpMYC65 gene and the key enzyme genes GpFPS1, GpSS1, GpSSE2, GpSE3, and GpOSC1 in the biosynthetic pathway of dammarane-type triterpenoid saponins were detected by qRT-PCR in overexpressing hairy roots (overexpressing lines OE1, OE2, OE3) and empty vector hairy roots (EV), with β-actin as the internal reference gene.

[0043] The primers used for qRT-PCR were as follows: GpMYC65-qRT-F (SEQ ID NO.17): 5'-CGTCGGACCCAAATGGCT-3'; GpMYC65-qRT-R (SEQ ID NO.18): 5'-ATCTGGCGTCGACGGTTG-3'; GpFPS1-qRT-F (SEQ ID NO.19): 5'-TAAATGTTCTTGGCTGGTCGTT-3'; GpFPS1-qRT-R (SEQ ID NO.20): 5'-ACGTTTGCTGGGTCTGCTTT-3'; GpSS1-qRT-F (SEQ ID NO.21): 5'-GAAAGGGTATCAGGAGGCGA-3'; GpSS1-qRT-R (SEQ ID NO.22): 5'-AACCTAGCCCGACAAGTCCA-3'; GpSE2-qRT-F (SEQ ID NO.23): 5'-GGAGGAGGAATTGCTGGTCTT-3'; GpSE2-qRT-R (SEQ ID NO.24): 5'-TGATTCTGCCTGCTCAAGTACC-3'; GpSE3-qRT-F (SEQ ID NO.25): 5'-GCCCAGCGGGTTTATGGT-3'; GpSE3-qRT-R (SEQ ID NO.26): 5'-GCTCCTCCCAGCCACATC-3'; GpOSC1-qRT-F (SEQ ID NO.27): 5'-TGGTGGTCTTAACGATGCTTGT-3'; GpOSC1-qRT-R (SEQ ID NO.28): 5'-TTCCAGCCCACTCATAAGCAC-3'.

[0044] The reaction system was as follows: SYBR Green qPCR Mix (2×), 5 μL; forward primer (10 μmol / L) for all target genes in this example, 0.3 μL; reverse primer (10 μmol / L), 0.3 μL; cDNA template, 2 μL; RNase-free ddH2O, 2.4 μL; total 10 μL.

[0045] The qRT-PCR reaction conditions are as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, for 40 cycles.

[0046] The results are as Figure 8 shown. In the overexpression lines OE1, OE2, and OE3, the expression levels of the GpMYC65 gene and the key enzyme genes GpFPS1, GpFPS1, GpSE2, and GpOSC1 in the dammarane-type triterpenoid saponin biosynthesis pathway were significantly higher than those in the EV. Among them, the expression level of the GpMYC65 gene in the overexpression lines was at least 60-fold higher than that in the EV line, the GpFPS1 gene in the overexpression lines was at least 20-fold higher than that in the EV line, the GpSS1 gene in the overexpression lines was at least 7-fold higher than that in the EV line, the GpSE2 gene in the overexpression lines was at least 8-fold higher than that in the EV line, and the GpOSC1 gene in the overexpression lines was at least 40-fold higher than that in the EV line. Thus, overexpression of the GpMYC65 gene can increase the expression of the key enzyme genes in the dammarane-type triterpenoid saponin biosynthesis pathway, especially promoting the expression of the GpOSC1 gene.

[0047] <Example 5> Determination of the content of dammarane-type triterpenoid saponins in hairy roots of Gynostemma pentaphyllum by targeted metabolomics: 1. Extraction of saponins from hairy roots of Gynostemma pentaphyllum (1) Fresh samples of Gynostemma pentaphyllum were placed in a freeze dryer (Scientz-100F) for vacuum freeze-drying; (2) Ground to a powder using a grinder (MM 400, Retsch) (30 Hz, 1.5 minutes); (3) Weighed 50 mg of sample powder using an electronic balance (MS105DΜ), and added 1200 μL of 70% methanol aqueous internal standard extraction solution pre-cooled to -20°C (if less than 50 mg, added according to the ratio of 1200 μL of extractant per 50 mg of sample); (4) Vortexed once every 30 minutes for 30 seconds each time, for a total of 6 times; (5) After centrifugation (at a speed of 12000 rpm for 3 minutes), the supernatant was aspirated, the sample was filtered through a microporous membrane (0.22 μm pore size), and stored in a sample vial for UPLC-MS / MS analysis.

[0048] 2. Chromatographic and mass spectrometric acquisition conditions The data acquisition instrument system mainly includes Ultra Performance Liquid Chromatography (UPLC) (ExionLC™ AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS).

[0049] The liquid phase conditions mainly include: 1) Chromatographic column: Agilent SB-C18 1.8 µm, 2.1 mm * 100 mm; 2) Mobile phase: Phase A is ultrapure water (added with 0.1% formic acid), and phase B is acetonitrile (added with 0.1% formic acid); 3) Elution gradient: The proportion of phase B is 5% at 0.00 min, linearly increases to 95% within 9.00 min, and is maintained at 95% for 1 min. From 10.00 - 11.10 min, the proportion of phase B drops to 5% and is balanced at 5% until 14 min; 4) Flow rate 0.35 mL / min; column temperature 40°C; injection volume 2 μL.

[0050] The mass spectrometry conditions mainly include: The temperature of the electrospray ionization (ESI) source is 500°C; the ion spray voltage (IS) is 5500 V (positive ion mode) / -4500 V (negative ion mode); the source gas I (GSI), gas II (GSII) and curtain gas (CUR) are set to 50, 60 and 25 psi respectively, and the collision-induced ionization parameters are set to high. QQQ scans use the positive ion multiple reaction monitoring (MRM) mode, and the collision gas (nitrogen) is set to medium. Through further optimization of the declustering potential (DP) and collision energy (CE), the DP and CE of each MRM ion pair are completed. According to the metabolites eluted in each period, a specific set of MRM ion pairs is monitored in each period.

[0051] 3. Qualitative and quantitative analysis The mass spectrometry data is processed using the software Analyst 1.6.3. Figure 9 Shown is the multi-peak diagram of MRM metabolite detection (ion current spectrum diagram extracted from multiple substances, XIC). The abscissa is the retention time (Rt) of metabolite detection, and the ordinate is the ion current intensity of ion detection (the intensity unit is cps, count per second).

[0052] Based on the local metabolic database, qualitative and quantitative analysis of metabolites in the samples was performed by mass spectrometry. Figure 9 The multi-peak graph of metabolite detection in the multiple reaction monitoring (MRM) mode shows the substances that can be detected in the samples. Each chromatographic peak of a different color represents a detected metabolite. Characteristic ions of each substance were screened out by a triple quadrupole, and the signal intensity (CPS) of the characteristic ions was obtained in the detector. The off-line mass spectrometry file of the sample was opened with MultiQuant software for integration and calibration of chromatographic peaks. The peak area (Area) of each chromatographic peak represents the relative content of the corresponding substance. Finally, all the integrated data of chromatographic peak areas were exported and saved.

[0053] To compare the differences in the content of each metabolite among all detected metabolites in different samples, according to the information on metabolite retention time and peak shape, as Figure 9 shown, we calibrated the chromatographic peaks of each metabolite detected in different samples to ensure accurate qualitative and quantitative analysis. As Figure 10 shown in A, after overexpressing GpMYC65, there was no obvious change in the appearance of the hairy roots of Gynostemma pentaphyllum. Compared with EV, the contents of gypenoside XVII, gypenoside VN2, gypenoside XXIII, and gypenoside Rd in the roots of three GpMYC65-overexpressing Gynostemma pentaphyllum hairy roots were significantly increased. Among them, gypenoside XVII had the highest content among the four saponin components, with an increase of more than 10 times; gypenoside XXIII increased by about 8 times on average. Gypenoside VN2 and gypenoside Rd were not detected in the EV hairy roots, but were significantly increased in the overexpression lines OE1, OE2, and OE3 ( Figure 10 B, 10C).

[0054] The above examples show that we cloned the transcription factor GpMYC65 from Gynostemma pentaphyllum leaves and constructed a vector for overexpressing GpMYC65. Using the genetic transformation method mediated by Agrobacterium rhizogenes (K599), transgenic hairy root systems overexpressing GpMYC65 were obtained. The experimental results showed that, compared with the control root systems, the expression levels of the key enzyme genes in the dammarane-type triterpenoid saponin synthesis pathway in the GpMYC65-overexpressing root systems were significantly increased. At the same time, the contents of the main Gynostemma pentaphyllum saponin components such as gypenoside XVII, gypenoside VN2, gypenoside XXIII, and gypenoside Rd in the GpMYC65-overexpressing root systems were also significantly increased.

[0055] The nucleotide sequence of the Gynostemma pentaphyllum transcription factor GpMYC65 is shown in SEQ ID NO.1: The amino acid sequence of the protein encoded by the Gynostemma pentaphyllum transcription factor GpMYC65 is shown in SEQ ID NO.2: MSYTFIVICYVFLVYSITVECWFLYSILLSLLQYRLTDSRKRLIYLFNYLPHLKQNNYSAVQLQLQLQMERLQAPILPNPNFNPCFHGEILEQSFAFEELEQAYFLTSTLESDQIPFLQMLESQPFKDPNFQTLLRLQHLNKPWEQEQEVNQIQELVELYSSPIKSEARYLNQHPNSASASASGLSSECNKNQPPHCCYSRRTQMAKTSPATKERRKRKRTRPAKNKEEVESQRMTHIAVERNRRRQMNDHLNVIKSLIPTSYVQRGDQASIIGGAIDFVKELEQLLESLEAQRKGEKGGCNGEYSPAPTSSSAMAMASNGLVMERRIGEGVCAEHKSEVAEIEVTLIQTHVNLKIKCPKRQGQLLKTIVALEDLRLTVLHLNITTSQATATMLYSFNLKMEDECKLGSAEQIAGKVHQILSFINES Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those skilled in the art, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the embodiments shown and described herein.

Claims

1. Gynostemma pentaphyllum transcription factor GpMYC65, characterized in that, The nucleotide sequence of the Gynostemma pentaphyllum transcription factor GpMYC65 is shown in SEQ ID NO.

1.

2. The protein encoded by the transcription factor GpMYC65 of Gynostemma pentaphyllum, characterized in that, The amino acid sequence of the protein encoded by the Gynostemma pentaphyllum transcription factor GpMYC65 is shown in SEQ ID NO.

2.

3. Recombinant expression vector, characterized in that, The recombinant vector contains the Gynostemma pentaphyllum transcription factor GpMYC65 described in claim 1.

4. The application of the Gynostemma pentaphyllum transcription factor GpMYC65 described in claim 1 in regulating the content of dammarane-type triterpenoid saponins in Gynostemma pentaphyllum.

5. The application according to claim 4, wherein The dammarane-type triterpenoid saponins are selected from at least one of gypenoside XVII, gypenoside VN2, gypenoside XXIII or gypenoside Rd.

6. The application according to claim 4, characterized in that It includes the following steps: Step 1: Construct an overexpression vector containing the coding gene of the transcription factor GpMYC65. Step 2: Use the Agrobacterium-mediated method to transform the overexpression vector into Gynostemma pentaphyllum leaves to obtain genetically transformed hairy roots, and screen and identify positive hairy roots overexpressing the transcription factor GpMYC65. Step 3: Carry out liquid culture on the screened positive hairy roots to obtain transgenic hairy roots with a dammarane-type triterpenoid saponin content higher than that of wild-type hairy roots.

7. The application according to claim 6, wherein, The method for constructing the overexpression vector in step 1 includes the following steps: A1: Using Gynostemma pentaphyllum leaf cDNA as a template and GpMYC65-F and GpMYC65-R as primers, obtain the GpMYC65 gene fragment by PCR amplification. A2: Connect the GpMYC65 gene fragment to the intermediate vector pDNOR221 and recombine it into the overexpression vector pK2GW7 to obtain an overexpression vector containing the coding gene of the transcription factor GpMYC65.

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