Panax notoginseng transcription factor gene pnhys and application thereof

Abstract

The application discloses a panax notoginseng transcription factor gene PnHY5 and application thereof in improving total saponin content and monomer saponin content of panax notoginseng cells, wherein the nucleotide sequence of the PnHY5 gene is shown as SEQ ID NO:1; the application proves that the panax notoginseng PnHY5 transcription factor has a function of positively regulating panax notoginseng saponin biosynthesis by using functional genomics and metabolic engineering related technologies; the panax notoginseng PnHY5 transcription factor gene is constructed on a plant expression vector and is transferred into panax notoginseng cells to make the panax notoginseng PnHY5 transcription factor gene overexpress, so that the expression amount of a key enzyme gene of a panax notoginseng saponin synthesis pathway is improved, and the biosynthesis of panax notoginseng total saponins is promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosynthesis, and particularly relates to a Panax notoginseng saponin biosynthesis related transcription factor gene PnHY5 and use thereof. BACKGROUND

[0002] Panax notoginseng is a medicinal plant of the Araliaceae family, and the roots and rhizomes thereof are used as traditional Chinese medicinal materials. Panax notoginseng has the dual effects of activating blood and stopping bleeding, and is an important component of the famous Yunnan Baiyao. Panax notoginseng saponins are the main active components of Panax notoginseng, and more than 70 dammarane-type tetracyclic triterpene saponins such as ginsenoside Rb1, Rg1, Rd and Re have been isolated and identified from the root tuber, rhizome (cutting), stem, leaf and flower of Panax notoginseng. Most of these monomer saponins are 20(S)-protopanaxadiol and 20(S)-protopanaxatriol, and no oleanolic acid-type saponin has been found, which is significantly different from ginseng and American ginseng. Panax notoginseng saponins have the effects of expanding coronary and peripheral blood vessels, increasing cerebral blood flow, inhibiting platelet aggregation, reducing blood viscosity, inhibiting thrombus formation, lowering blood lipids, resisting fatigue, resisting hypoxia, improving and enhancing the function of macrophages, etc.

[0003] Panax notoginseng is a perennial medicinal plant, and has high requirements for habitats, but the area of land suitable for planting Panax notoginseng is limited. Meanwhile, the growth of Panax notoginseng is seriously affected by diseases and insect pests, and the problems of soil secondary salinization and acidification are prominent, resulting in the need for crop rotation for Panax notoginseng planting. This long growth cycle and low land utilization rate have restricted the sustainable development of the Panax notoginseng industry. In recent years, the market for medicines containing Panax notoginseng has rapidly expanded, leading to an increase in demand for Panax notoginseng medicinal materials and a prominent contradiction between supply and demand. In view of the long time required for artificial cultivation and unclear chemical synthesis mechanism and route, the use of biological engineering technology and gene regulation methods to produce Panax notoginseng saponins has gradually become a research hotspot.

[0004] Light is the energy source of plant photosynthesis and one of the most important environmental factors in the process of plant growth and development. Plants have evolved a set of complex light sensing pathways to respond to changes in light environment during plant growth and development. These light sensing pathways can respond to light intensity, light quality, light direction and day length, etc. Among them, plants mainly perceive different wavelengths of light through different light receptors and transmit light signals. The sensing of light signals by light receptors can activate different families of transcription factors downstream of light receptors to transmit light signals. Currently, it has been reported that the families of transcription factors acting downstream of light receptors include bZIP, bHLH, MYB, Zinc-finger, GATA and GT1. These transcription factors transmit light signals mainly by binding to light response elements such as G-box, GATA, MYB, etc. on the promoters of downstream genes, thereby starting the transcription of downstream genes. HY5 belongs to the bZIP transcription factor family, and the protein encoded by HY5 is a core light signal regulator in the light signal transduction pathway downstream of light receptors, which plays a very important role in the process of light signal transduction and can directly regulate or regulate the expression of downstream genes by interacting with other transcription factors. HY5 can directly bind to the binding site containing the ACGT element on the promoter of the gene, such as G-box (CACGTG), A-box (TACGTA), Z-box (ATACGGT) and C-box (GACGTC). G-box widely exists in the promoters of light-controlled genes and some key metabolic pathway enzyme genes, has a highly conserved core motif CACGTG, is a general regulatory element for plant response to external environmental stress, and is also the most clear plant regulatory element in the field of plant research. As a transcription factor, HY5 exerts transcriptional regulation by first binding to the cis-element containing the ACGT element, and then controlling the expression of numerous target genes to respond to light signals, and ultimately regulating the physiological and biological processes of plants. HY5 can regulate many important aspects in the process of plant growth and development, such as cell elongation, cell proliferation, chloroplast development, pigment accumulation and nutrient assimilation. In recent years, studies have also reported the role of HY5 in other aspects, such as hormone signal transduction, synthesis of secondary metabolites, plant defense and response to external environment, etc. However, the role of HY5 transcription factor in metabolic regulation in Panax notoginseng is not clear. SUMMARY

[0005] The present application provides a Panax notoginseng transcription factor gene PnHY5, which is cloned from Panax notoginseng. The present application clarifies the use of the transcription factor, i.e. it can increase the expression amount of key enzyme genes in the synthesis metabolic pathway of notoginsenosides and increase the content of notoginsenosides in Panax notoginseng cells.

[0006] The nucleotide sequence of the transcription factor gene PnHY5 cloned from Panax notoginseng cells is shown as SEQ ID NO:1, the full-length cDNA sequence of the gene is 495 bp, and the protein encoded by the amino acid sequence shown as SEQ ID NO:2.

[0007] The transcription factor gene can be applied to the positive regulation of the biosynthesis of notoginsenosides, and the specific operation is as follows:

[0008] (1) Obtaining the full-length cDNA of the gene: specific primers for amplifying PnHY5 are used to extract total RNA from Panax notoginseng cells, and the coding region of PnHY5 is amplified by reverse transcription-polymerase chain reaction (RT-PCR), and then it is connected to the pMD18-T vector, and the cloning with the target gene is obtained by sequencing;

[0009] (2) Construction of plant expression vector and genetic transformation: the pMD18-T-PnHY5 plasmid is digested with restriction enzymes Xba I and Pst I, and the target gene fragment is obtained by gel recovery; the plant expression vector pCAMBIA2300S is digested with the same endonuclease, and the vector large fragment is obtained by gel recovery; the target gene fragment and the pCAMBIA2300S vector fragment are connected to construct the plant overexpression vector pCAMBIA2300S-PnHY5; the pCAMBIA2300S-PnHY5 plasmid is introduced into the Agrobacterium strain EHA105 by liquid nitrogen freeze-thaw method, and the PnHY5 is introduced into Panax notoginseng cells to make it overexpress, and the positive transgenic cell line is screened by antibiotic selection and fluorescent quantitative PCR;

[0010] (3) Detection of total saponin content in transgenic cell lines: saponins in Panax notoginseng non-transgenic cells and transgenic cell lines are extracted, and the difference in total saponin content between non-transgenic and transgenic cell lines is analyzed to screen the positive transgenic cell line with improved total saponin content;

[0011] (4) Detection of the content of part of important monomer saponins in transgenic cell lines: saponin solutions of Panax notoginseng non-transgenic cells and transgenic cell lines with improved total saponin content are prepared, and the content of part of important monomer saponins in non-transgenic and transgenic cell lines is determined by HPLC method, the difference in monomer saponin content between non-transgenic and transgenic cell lines is analyzed, and finally the positive transgenic cell line with improved monomer saponin content is screened.

[0012] The application takes in vitro cultured Panax notoginseng cells as a research object, clones PnHY5 transcription factor gene, and performs function identification on the transcription factor, so as to determine the position and role of the PnHY5 transcription factor in the saponin biosynthesis process of Panax notoginseng, and provide a theoretical reference and basis for obtaining efficient and stable saponin synthesis regulation technology and the establishment of a homologous or heterologous efficient expression system.

[0013] The application provides a new method for improving the content of saponin in Panax notoginseng, and the saponin in Panax notoginseng can be synthesized more efficiently by using the biological engineering technology and the gene regulation method, and the defects such as long artificial cultivation period, unclear chemical synthesis mechanism and route are overcome; the transcription factor PnHY5 gene is introduced into Panax notoginseng cells to express, so that the expression amount of the key enzyme gene in the saponin biosynthesis pathway of Panax notoginseng is increased, the yield of the saponin in Panax notoginseng is increased, and a theoretical reference and scientific basis are provided for large-scale industrial production of the saponin in Panax notoginseng. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is the amplification result of the full-length cDNA of PnHY5 in the application, wherein M is DL5000 DNA Marker, and 1 is the amplification product of the full-length cDNA of PnHY5;

[0015] Figure 2 It is the PCR detection result of the bacterial liquid of the plant expression vector pCAMBIA2300s-PnHY5 transferred into Agrobacterium tumefaciens EHA105 in the application, wherein Marker is DL5000 DNA Marker; 1-7 are bacterial liquid samples; the positive control is the PCR product with the plasmid pMD18-T-PnHY5 as a template; and the negative control is the PCR product with sterile water as a template;

[0016] Figure 3 It is the detection result of the resistance gene in the transgenic Panax notoginseng cells, wherein Maker is DL2000 DNA Maker; 1-3 are cDNA samples of the transgenic Panax notoginseng cells; the positive control is the PCR product with the plasmid pCAMBIA2300s-PnHY5 as a template; and the negative control is the cDNA of the non-transgenic Panax notoginseng cells.

[0017] Figure 4 It is the qRT-PCR result analysis diagram in the application, which shows the expression levels of the FPS, HMGR and DS genes in the saponin synthesis pathway of Panax notoginseng regulated by PnHY5 in the wild type and the transgenic cell lines, wherein WT is a wild type cell line, and 1-3 are transgenic cell lines;

[0018] Figure 5 It is the total saponin content determination result of the Panax notoginseng cells in the application, wherein WT is a wild type cell line, and 1-3 are transgenic cell lines;

[0019] Figure 6 The content determination results of some important monomer saponins in Panax notoginseng cells of the present application, wherein WT is a wild type cell line, and 1-3 are transgenic cell lines. DETAILED DESCRIPTION

[0020] The present application is further illustrated by the following examples, but these examples do not limit the scope of protection of the present application. The methods in the examples are all conventional methods, and the reagents used are all conventional commercially available reagents or reagents prepared according to conventional methods, unless otherwise specified.

[0021] Example 1: Cloning of PnHY5 gene fragment

[0022] Total RNA was extracted from Panax notoginseng cells (derived from Panax notoginseng callus), and the Panax notoginseng cells were ground into powder with liquid nitrogen, then transferred into a centrifuge tube, and total RNA was extracted by guanidine isothiocyanate method; the better quality RNA was selected to synthesize the first strand of cDNA using GoScript reverse transcription system of Promega company, Oligo(dT)15 1.0 μL, total RNA 5.0 μg, Nuclease-free Water was added to 10 μL, fully mixed gently, and placed in a 70℃ water bath for 5 min for pre-denaturation, then immediately placed on ice for 5 min, then added Nuclease-free Water 1.6 μL, GoScript TM 5×Reaction Buffer 4.0 μL, PCR Nucleotide Mix 1.0 μL, MgCl2(25 mM) 2.0 μL, Recombinant Ribonuclease Inhibitor 0.4 μL, GoScript TMReverse Transcriptase 1.0 μL mixed, centrifuged, 25°C annealing 5 min, 42°C water bath extension 90 min, finally, placed in 70°C water bath for 15 min, terminated reverse transcriptase activity, obtained Panax notoginseng cDNA first strand. With the synthesized first strand cDNA as template, amplified PnHY5, the used upper and lower primers were 5'-GGATCTTCCAGAGATTCTAGAATGCCAGACCCAGCAACAAG-3' and 5'-CTGCCGTTCGACGATCTGCAGTTATGTTTTGATAGGATGATTTGTGTAAA-3', respectively; PCR reaction conditions were 94°C 5 min, 94°C 30 s, 60°C 30 s, 72°C 30 s (32 cycles), 72°C 7 min; reaction system (20 μL) was 0.5 μL cDNA, 2 μL 10 x Advantage2 PCR Buffer, 0.4 μL 50 x dNTP Mix (10 mM each), 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 0.4 μL Advantage 2 PCR Polymerase Mix, 15.9 μL PCR-Grade water; after PCR, 5 μL was used for agarose gel electrophoresis to detect the specificity and size of the amplified product, and the target gene was amplified as shown in Figure 1 .

[0023] T-A cloning was performed using kit pMD18-T Vector System I (Promega, USA), reaction system and operation process were as follows: 8 μL PCR product was added with 1 μL pMD18-T Vector (50 ng / μL) and 1 μL 2 x Ligationsolution I successively, mixed, and then placed in 16°C for overnight reaction, and the ligation product was transformed into E. coli DH5α using heat shock transformation method. Positive clones were screened using LB solid medium containing ampicillin (Amp), and several single colonies were selected, shaken, and then identified using specific primers for amplifying PnHY5, and as a result, plasmid pMD18-T-PnHY5 was successfully constructed.

[0024] Example 2: Construction of plant overexpression vector

[0025] The SanPrep column plasmid DNA mini-extraction kit (Shanghai Genechem Co., Ltd., China) was used to extract the E. coli plasmid pMD18-T-PnHY5 inserted with the PnHY5 gene and the plant expression vector pCAMBIA2300s plasmid. 1 μL was used for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. The restriction endonucleases Xba I (TaKaRa, Japan) and Pst I (TaKaRa, Japan) were used for double enzyme digestion (50 μL system) of the plasmids pMD18-T-PnHY5 and pCAMBIA2300s. The reaction system and operation process were as follows: 20 μL pMD18-T-PnHY5 and pCAMBIA2300s plasmid was added with 7.5 μL 10×K buffer, 2.5 μL Xba I, 2.5 μL Pst I, and 17.5 μL ddH2O, mixed and centrifuged for a short time, and then placed in a 37°C enzyme for 3 h. All the enzyme digestion products were subjected to electrophoresis on an agarose gel, and then the PnHY5 fragment and the pCAMBIA2300s vector large fragment were recovered from the gel, respectively. The SanPrep column DNA gel recovery kit (Shanghai Genechem Co., Ltd., China) was used in the whole process. 1.0 μL of the recovered product was subjected to agarose gel electrophoresis to detect the size and concentration of the recovered fragment, and then stored at -20°C for later use.

[0026] The recovered PnHY5 DNA fragment and the pCAMBIA2300s vector fragment were connected using T4 DNA Ligase (TaKaRa, Japan). The reaction system (10 μL) and operation process were as follows: 6 μL PnHY5 DNA fragment was added with 2 μL pCAMBIA2300s vector DNA, 1 μL 10×T4 DNA Ligase Buffer, and 1 μL T4 DNA Ligase, mixed and centrifuged for a short time, and then subjected to an overnight reaction in a 16°C water bath. Then the heat shock transformation method was used to transform the ligation product into E. coli DH5α, and the solid medium containing 50 mg / L kanamycin (Kana) was used to screen positive clones. Single colonies were selected for shaking culture, and the bacterial solution was used as a template for PCR with specific primers for amplifying PnHY5. The clones with successful connection of PnHY5 and pCAMBIA2300s were selected. If the detected strain was positive, glycerol was added and stored at -80°C for later use.

[0027] The pCAMBIA2300s-PnHY5 plasmid in the above E. coli was extracted and purified. Then the constructed plant expression vector pCAMBIA2300s-PnHY5 was transformed into the Agrobacterium EHA105 competent cells by liquid nitrogen freezing and thawing method. The operation steps were as follows: 2 μg of pCAMBIA2300s-PnHY5 plasmid was added into a centrifuge tube containing 100 μL of competent cells, mixed gently, and then placed in ice bath for 5 min, followed by freezing in liquid nitrogen for 5 min, and then quickly placed in 37 °C water bath for 5 min, and then immediately placed in ice bath for 2 min, and then 800 μL of LB liquid medium was added and cultured at 28 °C for 4 h. The activated Agrobacterium was coated on LB solid medium containing 50 mg / L Kana and 20 mg / L Rif, and cultured at 28 °C. Single colonies were selected and cultured, and specific primers for amplifying PnHY5 were used for PCR to detect whether pCAMBIA2300s-PnHY5 was transformed into Agrobacterium. For the positive clones, glycerol was added and stored at -80 °C for later use. The PCR analysis results of pCAMBIA2300s-PnHY5 transformed into Agrobacterium EHA105 are shown in Figure 2. Figure 2 The plant overexpression vector pCAMBIA2300s-PnHY5 was successfully constructed.

[0028] Example 3: Agrobacterium-mediated plant genetic transformation and screening of transgenic plants

[0029] The Agrobacterium EHA105 strain containing pCAMBIA2300s-PnHY5 plasmid stored in the -80 °C refrigerator was inoculated in 5 mL of LB liquid medium containing 50 mg / L Kana and 25 mg / L rifampicin, and cultured at 28 °C until turbid. 1 mL of turbid bacterial solution was taken and coated on LB solid medium containing 50 mg / L Kana, and cultured at 28 °C for 48 h. The Agrobacterium on the LB solid medium was scraped and inoculated in MGL liquid medium with the addition of 50 mg / L acetosyringone, and cultured at 28 °C until the OD 600 was 0.6, and then the bacterial solution was used to dip the Panax notoginseng cells.

[0030] The well-growing Panax notoginseng cells were inoculated into MS pre-culture medium (50 mg / L acetosyringone) for pre-culture for 3 days. After pre-culture, the cells were completely immersed in the above-mentioned Agrobacterium liquid for shaking culture. After immersion and infection, the bacterial liquid was removed, the bacterial liquid on the surface of the cells was absorbed with sterile filter paper, and then the cells were inoculated into MS co-culture medium (containing 50 mg / L acetosyringone) for co-culture for 3 days. The Panax notoginseng cells after co-culture were carefully transferred into an antibiotic-free medium (adding cephalosporin to the Panax notoginseng cell culture medium, the Panax notoginseng culture medium is MS medium + 2,4-D 4 mg / L + KT 1 mg / L + 6-BA 0.5 mg / L, pH 5.6), and the content of cephalosporin in the antibiotic-free medium was 200 mg / L, 100 mg / L and 50 mg / L, respectively. In order from high to low concentration, the antibiotic-free medium was placed for 3 days in turn until no Agrobacterium grew on the surface of the medium. After 9 days of antibiotic-free culture, the Panax notoginseng cells were transferred to a screening medium (adding neomycin to the Panax notoginseng cell culture medium to a final concentration of 50 mg / L), and subcultured once about 35 days. After 4-5 times of subculture and screening, a transgenic Panax notoginseng cell line was obtained.

[0031] The genomic DNA of the transgenic Panax notoginseng cell line was extracted by the improved CTAB method, and the upstream and downstream primers npt F (5'-CTCTGATGCCGCCGTGTT-3') and npt R (5'-CCCTGATGCTCTTCGTCCA-3') were designed according to the kanamycin npt II resistance gene sequence on the T-DNA in the pCAMBIA2300s vector. PCR detection was performed with the extracted Panax notoginseng genomic DNA as a template to screen positive transgenic Panax notoginseng cells.

[0032] The results are shown in Figure 3 , and a specific band of about 0.4 kb in size was amplified in the figure, which was consistent with the expected size, indicating that the three Panax notoginseng transgenic cell lines had introduced exogenous DNA and integrated into the genomic DNA and stably inherited, and the PnHY5 gene transgenic Panax notoginseng cell line was preliminarily determined.

[0033] Example 4: Effect of PnHY5 gene overexpression on the expression amount of key enzyme genes FPS, HMGR and DS in Panax notoginseng saponin synthesis pathway

[0034] RNA was extracted from Panax notoginseng transgenic cell lines and wild-type cell lines that were approximately 25 days old and in good growth condition. The first strand of cDNA was synthesized using the Promega GoScript reverse transcription system. The reaction system and procedure were as follows: High-quality RNA was used to synthesize the first strand of cDNA using the Promega GoScript reverse transcription system. Oligo(dT) 151.0 μL, Total RNA 5.0 μg, and Nuclease-free Water were added to a final volume of 10 μL and thoroughly mixed. The mixture was then placed in a 70°C water bath for 5 min for pre-denaturation, followed immediately by an ice bath for 5 min. The following reagents were then added: Nuclease-free Water 1.6 μL, GoScript™ 5× Reaction Buffer 4.0 μL, PCR Nucleotide Mix 1.0 μL, MgCl2 (25 mM) 2.0 μL, and Recombinant... Ribonuclease Inhibitor 0.4μL, GoScript TM Mix 1.0 μL of reverse transcriptase, centrifuge briefly, anneal at 25°C for 5 min, extend in a 42°C water bath for 90 min, and finally place in a 70°C water bath for 15 min to terminate reverse transcriptase activity, thus obtaining the first strand of Panax notoginseng cDNA.

[0035] Using this cDNA as a template, primers were designed based on the following genes: Panax notoginseng GAPDH gene (accession number: KF815711.1), Farnesyl pyrophosphate synthase (FPS) gene (accession number: DQ059550.1), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene (accession number: KJ578757.1), and dammarenediol-II synthase (DS) gene (accession number: KC953035.1). Follow the instructions in the qPCR Master Mix manual; the primer sequence used is GAPDH. F :5'-CTACCAACTGTCTTGCTCCCCT-3';GAPDH R :5'-TGATGCAGTCTTCCACCTCTC-3';FPS F :5'-CGGATGCTGGACTATAATGTG-3';FPS R: 5'-ACACCCAGAAGGTTCAAGCAA-3'; DS F : 5'-GGCAGGACCCAGCACAAAATA-3'; HMGR R : 5'-ACACCCAGAAGGTTCAAGCAA-3'; DS F : 5'-TATGAGTGGGAAGGGTGC-3'; DS R : 5'-TGGCGATAATTGCTTGAGTA-3'. The specific reaction system and operation process are as follows: 20 ng of cDNA, 25 μL of qPCR Master Mix (2x), and 0.2 μL of qPCR Primers (GAPDH / GAPDH F / FPS R / FPS F / HMGR R / HMGR F / DS R / DS F / DS R , 10 mM) are added in a PCR tube, and the volume is made up to 50 μL with Nuclease-Free Water; the reaction system is vortexed and centrifuged to collect at the bottom of the tube, and then it is placed in a fluorescent quantitative PCR instrument for reaction, and the fluorescent quantitative PCR is performed by two-step method, and the reaction parameters are as follows: hot start 95 °C for 2 min; denaturation 95 °C for 15 s, annealing / extension 60 °C for 1 min, a total of 45 cycles, and each sample is repeated 2 times for each gene.

[0036] The qRT-PCR results show that the expression amounts of the FPS, HMGR, and DS genes in the PnHY5 gene transformed Panax notoginseng cells are significantly higher than those in the wild type ( Figure 4 ), indicating that PnHY5 as a transcription factor can promote the expression of the FPS, HMGR, and DS genes which are key enzymes in the saponin synthesis metabolic pathway of Panax notoginseng.

[0037] Example 5: Effect of PnHY5 gene overexpression on the synthesis amount of total saponins of Panax notoginseng

[0038] The transgenic cell line and wild type cell line grown for about 35 days were selected and placed in washed 100 mL triangular flask, 20 mL methanol solution was added and soaked overnight, then treated with ultrasonic wave at room temperature for 1 h, filtered, the filtrate was collected, concentrated, then dissolved with methanol, and diluted to 25 mL to obtain the crude extract; the residue was dried at 50°C to constant weight, and weighed. 5 mL of the crude extract was precisely taken and placed in 50 mL beaker, and evaporated to dryness in water bath; after evaporation, it was dissolved with 4 times volume of distilled water, and after fully dissolved, filtered, and the filtrate was transferred to the treated Hsp100 macroporous resin column, and 2 column volumes of distilled water were slowly washed to remove sugar and other impurities. Whether the sugar impurities were removed completely was detected by Molish reaction, if the result was positive, continue to wash with distilled water until negative, then elute with 2 column volumes of 75% ethanol solution, collect the alcohol, evaporate to dryness in water bath, and dissolve the residue with methanol solution, and dilute to 25 mL.

[0039] Precisely take 150 μL of the above sample into a 10 mL test tube (3 replicates), evaporate the solvent, add 0.2 mL of freshly prepared 5% vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid, mix well, heat in 60°C water bath for 15 min, immediately cool with ice water, add 5 mL of glacial acetic acid, mix well, stand for 10 min, then measure the absorbance at 554 nm, and calculate the PNS content according to the standard curve. The results show that the total saponin content in the PnHY5 gene-transferred Panax notoginseng cells is higher than that in the wild type cells Figure 5 ), combined with the qRT-PCR results, it is indicated that the PnHY5 transcription factor is involved in the synthesis and metabolic regulation of notoginseng saponins, and helps to improve the saponin yield.

[0040] Example 6: Effect of PnHY5 gene overexpression on the synthesis amount of notoginseng monomer saponins

[0041] The contents of some important monomer saponins (Rb1, Rg1, Rd, Re) in Panax notoginseng cell lines were determined by HPLC method. The high performance liquid chromatography conditions were as follows: high performance liquid chromatograph, Waters symmertry C18 chromatographic column (4.6×250 mm, 5 μm), linear gradient elution (v / v) was carried out with acetonitrile (A): water (B) as mobile phase, column temperature was 30°C, detection wavelength was 203 nm, and flow rate was set to 1.0 mL / min.

[0042] Accurately weigh a certain amount of monomer ginsenoside Rb1, Rg1, Rd, Re standard substance, add 1 mL of methanol solution, and prepare a standard substance mixed solution with concentrations of 340, 300, 260, 320, 280, 300 μg / mL, respectively. Inject 4, 6, 8, 10, 15, 20, 25, 30 μL of the mixed standard substance solution into the high performance liquid chromatograph, respectively. Take the injection amount (μg) as the abscissa (x) and the area of the chromatographic peak as the ordinate (y) to draw the linear regression equation of each monomer ginsenoside.

[0043] Respectively weigh 0.1 g of non-transgenic and transgenic Panax notoginseng cell powder with improved total ginsenoside content in a clean 50 mL triangular flask, and add 10 mL of 70% methanol solution to each. After overnight soaking, treat with an ultrasonic disrupter for 90 min (60 w, 4 s / 5 s). After ultrasonic treatment, remove the residue and leave the filtrate, and place the filtrate in a 50°C oven overnight to dry. Add 10 mL of distilled water to dissolve the dried residue, and then extract with the same volume of water-saturated n-butanol (do not use n-butanol that has not been water-saturated) 2-3 times, and place the final collected extract in a 50°C oven overnight to dry. Dissolve the dried residue with an appropriate amount of 100% methanol, and then dilute the solution to 5 mL, mix uniformly, and filter through a 0.45 μm filter membrane. Determine the content of some important monomer ginsenosides in the ginsenoside solution by high performance liquid chromatography. The results show that Rb1, Rg1, Rd, and Re monomer ginsenosides are detected in both non-transgenic Panax notoginseng cells and transgenic Panax notoginseng cell lines, and the four monomer ginsenosides in the transgenic cell line increase by different magnitudes compared with the non-transgenic cell line. Figure 6 )

Claims

1. A Panax notoginseng transcription factor gene, the nucleotide sequence of which is shown as SEQ ID NO:

1. PnHY5 2. The Panax notoginseng transcription factor gene of claim 1, wherein the nucleotide sequence of the gene is shown as SEQ ID NO:

1.

2. The panax transcription factor gene of claim 1 PnHY5 The application is used in improving total saponin content and monomer saponin content of panax cells, and is characterized in that: The monomer saponin is Rb1, Rg1, Rd and Re.

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