A gene for regulating ginsenoside content, PgLBD45, and its application.

Abstract

This invention discloses a gene derived from ginseng. PgLBD45 The gene was overexpressed and its application in increasing ginsenoside content was studied. Overexpression of this gene can effectively regulate the ginsenoside content in ginseng roots. This invention detected nine monomeric saponins, and the results showed that five of the obtained saponins... PgLBD45 The contents of Rh1 and Rb2 in overexpressing positive hairy roots were significantly decreased, while the content of Rb1 was significantly increased. These results indicate that... PgLBD45 Genes are involved in regulating the synthesis of ginsenosides. This invention utilizes overexpression... PgLBD45 Genes can significantly regulate the content of multiple ginsenosides, which is of great research value for studying ginsenoside content and provides a powerful technical means for developing ginseng germplasm resources with high ginsenoside content.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for regulating ginsenoside content. PgLBD45 Genes and their applications. Background Technology

[0002] Ginseng, a perennial herb belonging to the genus Panax of the Araliaceae family, is a precious traditional Chinese medicine with a long history of medicinal use in China. Modern medical research has found that ginsenosides, important secondary metabolites of ginseng, have therapeutic effects on various diseases. Based on the different aglycones they contain, ginsenosides can be roughly divided into oleanolic acid type and dammarane type, with the dammarane type further including protopanaxadiol and protopanatriol type saponins.

[0003] Ginsenosides are important secondary metabolites in ginseng and have extremely high economic value. According to the 2020 edition of the Pharmacopoeia of the People's Republic of China, ginsenosides Re, Rb1, and Rg1 are important evaluation indicators for ginseng quality control, demonstrating the significant role of saponins in evaluating ginseng quality.

[0004] Meanwhile, with the continuous development of medicine, people have discovered that ginsenosides have a wide range of pharmacological effects. Currently, the most in-depth research focuses on their anti-diabetic properties. Re can promote the peroxisome proliferator-activated receptor-γ (PPAR-γ) and its response genes. ADIPOQ , IRS1 and AP2 Resin (Re) regulates blood glucose levels by promoting fat formation. Furthermore, it can promote the translocation of GLUT4 from intracellular to the cell membrane, enhancing glucose uptake and processing in 3T3-L1 adipocytes. GLUT4, through conformational changes, brings glucose into the cell, thus helping to maintain stable blood glucose levels. Researchers have also found that Re can improve the efficacy of chemotherapy and reduce its toxicity. While the molecular mechanisms of its antidiabetic effects are not as thoroughly understood, it does reduce the levels of inflammatory markers in tissues and improve histopathological changes. Simultaneously, Re's immunomodulatory activity makes it a valuable adjuvant in vaccines, significantly enhancing immune efficiency.

[0005] The importance of ginsenosides is self-evident, both for the agricultural production and sales of ginseng itself and for its broad medicinal prospects. Therefore, elucidating the regulatory network of ginsenoside synthesis has become a key research focus. There are two main synthetic pathways for ginsenosides: the mevalonic acid pathway and the methyl erythritol pathway. Key enzyme genes functioning in these pathways have been continuously cloned and their functions verified in recent years, gradually clarifying the synthetic pathways. However, these pathways are not simply catalytic relationships but involve complex regulatory networks.

[0006] This study will identify genes highly related to ginsenoside synthesis from multiple levels and perspectives by examining the relationship between traits and gene expression, gene interactions, and the effects of gene mutations on traits, and will use overexpression to verify gene function. Summary of the Invention

[0007] This invention provides a method for regulating ginsenoside monomers. PgLBD45 Genes and their applications provide a powerful technical means for developing ginseng germplasm resources with high ginsenoside content.

[0008] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0009] This invention provides a ginseng that can regulate ginsenoside monomers. PgLBD45 Genes, the ginseng PgLBD45 The gene sequence is shown in SEQ ID NO.1, and the OFR sequence is shown in SEQ ID NO.2.

[0010] This invention also provides a method for amplifying ginseng. PgLBD45 The primer pairs for the gene, the base sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0011] Furthermore, the present invention also provides a ginseng containing the above-mentioned ginseng. PgLBD45 The carrier of genes.

[0012] Furthermore, the present invention also provides the aforementioned ginseng. PgLBD45 Application of genes in regulating the content of ginsenoside monomers.

[0013] The present invention has the following beneficial effects:

[0014] This invention discloses a gene derived from ginseng. PgLBD45 The gene was overexpressed and its application in increasing ginsenoside content was studied. Overexpression of this gene can effectively regulate the ginsenoside content in ginseng roots. This invention detected nine monomeric saponins, and the results showed that five of the obtained saponins... PgLBD45 The contents of Rh1 and Rb2 in overexpressing positive hairy roots were significantly decreased, while the content of Rb1 was significantly increased. These results indicate that... PgLBD45 Genes are involved in regulating the synthesis of ginsenosides. This invention utilizes overexpression... PgLBD45 Genes can significantly regulate the content of multiple ginsenosides, which is of great research value for studying ginsenoside content and provides a powerful technical means for developing ginseng germplasm resources with high ginsenoside content. Attached Figure Description

[0015] Figure 1 Electrophoresis diagram of total RNA from ginseng adventitious roots.

[0016] Figure 2 Electrophoresis diagram of total RNA reverse transcription to cDNA from ginseng adventitious roots.

[0017] Figure 3 : PgLBD45 Electrophoresis diagram of gene PCR products.

[0018] Figure 4 PCR electrophoresis image of Escherichia coli transformed with recombinant vector.

[0019] Figure 5 PCR electrophoresis image of Agrobacterium C58C1 bacterial culture transformed with recombinant vector.

[0020] Figure 6 : PgLBD45 Gene overexpression induces ginseng hairy root induction (A: ginseng adventitious root pre-culture; B: with) PgLBD45 C: Co-culture of Agrobacterium-mediated superexpression; D: Hairy root production;

[0021] Figure 7 :: PgLBD45 Overexpression of hairy roots for large-scale culture.

[0022] Figure 8 Overexpression PgLBD45 Electrophoresis diagram of PCR products from the genome of ginseng hairy root (a: Rol C gene; b: PgLBD45 downstream vector sequence of gene; c: containing PgLBD45 Partial vector sequence of the gene; d: PgLBD45 (Upstream vector sequence of the gene).

[0023] Figure 9 Overexpression in hair roots PgLBD45 Relative gene expression levels.

[0024] Figure 10 : PgLBD45 Detection of nine monomeric saponins in overexpression-positive hairy roots (Re, Rf, Rg1, Rg2, and Rh1 are protopanaxadiol-type saponins; Rb1, Rb2, Rg3, and Rh2 are protopanaxadiol-type saponins, with different color schemes used to distinguish the two types of saponins). "*" indicates... p ≤ 0.05, “**” represents p ≤ 0.01, “***” represents p ≤ 0.001). Detailed Implementation

[0025] Source of materials

[0026] The ginseng adventitious root material was provided by the Ginseng Resource Utilization and Research Laboratory of the Science and Engineering Building of Jilin Agricultural University.

[0027] The ginseng hair-like root material was obtained by infecting Agrobacterium C58C1 strain.

[0028] Main content

[0029] 1. Identification of genes related to the regulation of ginsenoside biosynthesis

[0030] Based on the Jilin ginseng transcriptome database, this study used differential expression analysis, correlation analysis, SNP / InDels mutation association analysis, weighted co-expression network analysis, and methyl jasmonic acid-induced expression analysis to obtain a gene population highly associated with ginsenosides. Combined with GO functional annotation results, one gene was ultimately identified as belonging to... LBD The genes in the gene family were selected as the subjects of subsequent research and named PgLBD45 .

[0031] 2. Extraction and reverse transcription of total RNA from ginseng

[0032] RNA was extracted from ginseng adventitious roots using the TransZol RNA Extraction Kit (Beijing). The extracted RNA was subjected to agarose gel electrophoresis (Figure 1). First-strand cDNA was synthesized using the SPARKscript II RT Plus Kit (WithgDNA Eraser) reverse transcription kit (Shandong Cisco). The product was examined by agarose gel electrophoresis (Figure 2).

[0033] 3. PgLBD45 Gene ORF full-length clone

[0034] Table 1. PgLBD45 Gene length information

[0035]

[0036] According to the primers described in SEQ ID NO. 3 and SEQ ID NO. 4 and the above PgLBD45 Gene sequence information, using the cDNA obtained by reverse transcription in point 2 above as the first-strand template, for... PgLBD45 PCR amplification was performed, and the PCR products were verified by agarose gel electrophoresis (Figure 3). Purification was then carried out using the SanPrep column-based PCR product purification kit from Sangon Biotech. The purified product was then ligated into Baori Biopharmaceutical pMD. TM Escherichia coli was transformed with the 18-T cloning vector.

[0037] 4. Construction of overexpression vectors

[0038] The overexpression vector used in this study was pCAMBIA1300-35S-sGFP (stored in the laboratory).

[0039] ①Use pCAMBIA1300-35S-sGFP vector Sac I and Xba I. Perform linearized double enzyme digestion;

[0040] ② Use primer pairs containing homologous arms for PgLBD45-pMD TM The 18-T recombinant vector was amplified by PCR to obtain a sample containing a homologous arm. PgLBD45 Gene;

[0041] ③ Homologous recombination was performed on the linearized vector and homologous arm gene fragments using the Beijing TransGen Basic Seamless Cloning and Assembly Kit;

[0042] ④ The recombinant product was transformed into Escherichia coli DH5α competent cells and cultured overnight on LB solid medium containing Kan resistance (vector determined);

[0043] ⑤ Pick a single colony and culture it in 1 ml of liquid LB medium containing kanamycin (50 µg / ml);

[0044] ⑥ Perform PCR amplification verification on the bacterial culture (Figure 4). After verification, send it to Sangon Biotech for sequencing verification.

[0045] 5. Preparation of Agrobacterium C58C1 engineered strain

[0046] ① In a clean bench, aspirate 5 µl of recombinant plasmid into competent cells, mix well, and place on ice for 5 min;

[0047] ②Prepare liquid nitrogen in advance, immerse the centrifuge tubes in liquid nitrogen for quick freezing for 5 minutes, and then place them in a metal bath (37℃) for heat shock for 5 minutes;

[0048] ③ In a clean bench, add 700 µl of liquid LB medium (without antibiotics) to a centrifuge tube and incubate at 28 ℃ and 170 rpm for 4 h with shaking.

[0049] ④ Remove the centrifuge tube and centrifuge at 4000 rpm for 3 minutes.

[0050] ⑤ After discarding the supernatant, resuspend the sample in LB liquid medium (approximately 100-200 µl), spread it on LB agar plates containing rifampicin and kanamycin (carrier-determined), and incubate in the dark at 28 °C for 48 h.

[0051] ⑥ Pick a single colony from the plate and inoculate it into 1 ml of LB liquid medium containing rifampicin and kanamycin (vector-determined) and culture overnight for bacterial PCR verification (Figure 5).

[0052] 6. Agrobacterium-mediated transformation of ginseng adventitious roots

[0053] ①Pre-culture

[0054] Healthy ginseng adventitious root segments were inoculated into MS solid-based culture medium containing 500 µl of hormone 1 and 50 µl of hormone 2, and cultured at 23 °C in the dark for 2 days.

[0055] ② Co-cultivation

[0056] Agrobacterium-mediated amplification of the recombinant overexpression vector to OD200. 600 The bacterial cells were collected by centrifugation at a concentration of 0.43. The bacterial culture was resuspended in 1 / 2 MS liquid medium containing acetylsyringone and activated at 28°C and 50 rpm for 1 h. The pre-cultured adventitious root segments were placed in the bacterial culture and incubated at 28°C and 80 rpm for 15 min. The bacterial culture was then dried, and the root segments were placed in 1 / 2 MS solid medium containing acetylsyringone and incubated at 23°C in the dark for 48 h.

[0057] ③ Sterilization culture

[0058] The co-cultured adventitious roots were placed on filter paper to absorb the bacterial solution, and then inoculated onto 1 / 2 MS solid medium containing cephalosporin. They were incubated at 23 °C in the dark until hairy roots emerged. The hairy roots that emerged could still propagate on hormone-free 1 / 2 MS solid medium before being used for subsequent positive verification (Figure 6).

[0059] ④ Propagation and cultivation

[0060] Hairy roots that could be stably subcultured on 1 / 2 MS solid medium were quantitatively added to 1 / 2 MS liquid medium for propagation culture. The culture conditions were dark, 22 ℃, and 110 rpm (Figure 7).

[0061] 7. Identification of positive hair roots

[0062] For the aforementioned hairy roots exhibiting hormone autotrophic properties, genomic DNA was extracted using the CTAB method to verify whether the T-DNA region of the overexpression vector was successfully inserted into the plant genome. PCR amplification was performed using the genomic DNA of a single hairy root system as a template, and the amplification results were verified using agarose gel electrophoresis (Figure 8). Single root systems matching the expected length were identified as... PgLBD45 Positive-positive plant material.

[0063] 8. Quantitative fluorescence verification of positive materials

[0064] Use the method described in point 2 to PgLBD45RNA was extracted from positive hair roots and positive hair roots transferred only with the pCAMBIA1300-35S-sGFP empty vector. Reverse transcription was performed using the Kangrun Bio StarScript II First-strand cDNASynthesis Mix With gDNA Remover reverse transcription kit. PgCYP As an internal reference gene, its expression level was verified using Applied Biosystems™ 7500 real-time quantitative PCR (Figure 9). PgLBD45 A single-root system exhibiting significant changes in gene expression is identified as... PgLBD45 Overexpression of positive hair roots.

[0065] 9. Extraction of saponins from positive hair-like roots

[0066] Dry the positive hair-like roots propagated by liquid, weigh 1 g of dry weight, grind them into powder, place them in 300 mL of distilled water, heat and concentrate until 1 / 3 of the volume remains, then stop heating, filter and collect the filtrate. Repeat 3 times, mix the filtrates, and add them to an ODS column that has been fully activated and equilibrated.

[0067] ① After all the leaching solutions have passed through the ODS column, add 50 mL of 20% methanol to elute impurities, and repeat 3 times.

[0068] ② After eluting impurities, add 50 mL of 100% methanol to elute saponins. Repeat 3 times and collect the eluent in a rotary evaporator.

[0069] ③ After all the methanol has evaporated, crystals will remain. Use 5 mL of chromatographic methanol to reconstitute them.

[0070] ④ Filter the solution into the sample bottle using an organic filter (0.22 µm) to obtain the ginseng adventitious root saponin extract.

[0071] 10. Detection of positive hair root saponins

[0072] Nine ginsenoside monomeric standard solutions, mixed standard solutions, and extracted saponin sample solutions were analyzed by high-performance liquid chromatography (HPLC). The HPLC system was a Waterse2695, and the column was a Waters C18 column. The mobile phase consisted of chromatographic acetonitrile (A) and Wahaha purified water (B). Elution conditions are shown in Table 2. The injection volume was 10 µL, the column temperature was 30℃, and the mobile phase flow rate was 1.0 ml / min. The detection wavelength was 203 nm.

[0073] Table 2. HPLC detection time and mobile phase composition ratio

[0074]

[0075] Calculation formula:

[0076]

[0077]

[0078] Finally, Welch's t-test (without the assumption of homogeneity of variance) was used to compare the saponin data of each superphenomenon with the saponin data of the positive control single root system in two independent samples. Significance was marked with "*" in the figure. Figure 10 As shown.

[0079] The results showed that 5 PgLBD45 The contents of Rh1 and Rb2 in overexpressing positive hairy roots were significantly decreased, while the content of Rb1 was significantly increased. These results indicate that... PgLBD45 Genes are involved in regulating the synthesis of ginsenosides.

Claims

1. A ginseng method for regulating ginsenosides PgLBD45 Genes are characterized by: The ginseng PgLBD45 The gene sequence of the gene is shown as SEQ ID NO. 1, and the OFR sequence is shown as SEQ ID NO.

2.

2. A composition comprising the ginseng of claim 1 PgLBD45 a vector comprising the gene of claim 1.

3. The ginseng of claim 1 PgLBD45 application of the ginseng gene in regulating content of ginsenoside monomer.

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