PgLBD45 gene for regulating ginsenoside content and application of PgLBD45 gene

Abstract

The invention discloses a gene from ginseng, PgLBD45 and application of PgLBD45 in increasing ginsenoside. The content of ginsenoside at the root of ginseng can be effectively regulated and controlled by over-expression of the gene. According to the present invention, nine monomer saponins are detected, the results show that the contents of Rh1 and Rb2 of 5 PgLBD45 overexpression positive hairy roots obtained through the experiment are significantly reduced, the content of Rb1 is significantly increased, and the results show that the PgLBD45 gene participates in the regulation of the synthesis of ginsenoside. The over-expression of the PgLBD45 gene can significantly adjust the content of multiple ginsenosides, has an important research value on the research of the content of ginsenosides, and provides a powerful technical means for developing ginseng germplasm resources with high ginsenoside content.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to a method for regulating the content of ginsenosides. PgLBD45 Genes and their applications. Background Art

[0002] Ginseng, a perennial herb of the genus Panax, belongs to the Araliaceae family. It is a precious Chinese herbal medicine with a long history of medicinal use in China. Modern medical research has revealed that ginsenosides, important secondary metabolites of ginseng, have therapeutic effects on a variety of diseases. Ginsenosides can be broadly divided into oleanolic acid-type and dammarane-type saponins, depending on the aglycone they contain. Dammarane-type saponins further include protopanaxadiol and protopanaxatriol saponins.

[0003] Ginsenosides are important secondary metabolites of ginseng and possess 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 key indicators for ginseng quality control, demonstrating the crucial role of saponins in evaluating ginseng quality.

[0004] At the same time, with the continuous development of medicine, people have found that ginsenosides have a wide range of pharmacological effects. The most in-depth research now is on their anti-diabetic effects. Re can promote the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and its response genes. ADIPOQ 、 IRS1 and AP2 The expression of Re can regulate blood sugar by promoting the formation of fat. In addition, Re can also promote the translocation of GLUT4 from the intracellular to the cell membrane, enhance the uptake and disposal of glucose by 3T3-L1 adipocytes. GLUT4 changes its own conformation and takes glucose into the cell, thereby helping to maintain blood sugar stability. Later, researchers also found that Re has the effect of improving the efficacy of chemotherapy and reducing toxicity. Although it is not as thorough as the study of the molecular mechanism of anti-diabetes, it can indeed reduce the level of tissue inflammatory factor indicators and improve tissue pathological changes. At the same time, Re has immunomodulatory activity, and its use as a vaccine adjuvant can greatly improve the immune efficiency.

[0005] The importance of ginsenosides is self-evident, both for the agricultural production and sales of ginseng itself and for its broader medicinal potential. Deciphering the regulatory networks underlying ginsenoside synthesis has become a key research focus. Ginsenoside synthesis involves two primary pathways: the mevalonate pathway and the methylerythritol pathway. Key enzyme genes responsible for these pathways have been cloned and functionally verified in recent years, and the synthesis pathways are becoming increasingly clear. However, this involves more than a simple catalytic relationship; a complex regulatory network exists.

[0006] This study will obtain genes that are highly correlated with ginsenoside synthesis from multiple levels and angles through the relationship between traits and gene expression, the interaction between genes, and the impact of gene mutations on traits, and verify gene function using overexpression. Summary of the Invention

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

[0008] In order to achieve the above objectives, the technical solutions provided by the present invention are as follows: The present invention provides a ginseng capable of regulating ginsenoside monomers PgLBD45 gene, the ginseng PgLBD45 The gene sequence is shown in SEQ ID N0.1, and the OFR sequence is shown in SEQ ID N0.2.

[0009] The present invention also provides a method for amplifying ginseng PgLBD45 The primer pair of the gene, the base sequence of the primer pair is shown in SEQ ID N0.3 and SEQ ID N0.4 Furthermore, the present invention also provides a method comprising PgLBD45 Gene carrier.

[0010] Furthermore, the present invention also provides the above-mentioned ginseng PgLBD45 Application of genes in regulating the content of ginsenoside monomers.

[0011] The present invention has the following beneficial effects: The present invention discloses a gene from ginseng, PgLBD45 , and its application in increasing ginsenosides, overexpression of this gene can effectively regulate the content of ginsenosides in ginseng roots. The present invention detected a total of 9 monomer saponins, and the results showed that 5 of the experimental PgLBD45 The contents of Rh1 and Rb2 in the overexpression positive hairy roots decreased significantly, while the content of Rb1 increased significantly. PgLBD45 The gene is involved in regulating the synthesis of ginsenosides. PgLBD45 The gene can significantly regulate the content of multiple ginsenosides, which is of great research value for studying the content of ginsenosides and provides a powerful technical means for developing ginseng germplasm resources with high content of ginsenosides. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : Electrophoresis of total RNA from ginseng adventitious roots.

[0013] Figure 2 : Electrophoresis of total RNA reverse transcribed cDNA from ginseng adventitious roots.

[0014] Figure 3 : PgLBD45 Electrophoresis of gene PCR products.

[0015] Figure 4 : PCR electrophoresis of Escherichia coli transformed with recombinant vector.

[0016] Figure 5 : PCR electrophoresis of Agrobacterium C58C1 culture transformed with recombinant vector.

[0017] Figure 6 : PgLBD45 Gene overexpression induced ginseng hairy roots (A: ginseng adventitious root pre-culture; B: PgLBD45 Co-culture of overexpressing Agrobacterium; C: Production of hairy roots; D: Subculture of hairy roots).

[0018] Figure 7 :: PgLBD45 Overexpression of hairy roots was expanded and cultured.

[0019] Figure 8 : Overexpression PgLBD45 Electrophoresis of PCR products of ginseng hairy root genome (a: Rol C gene; b: PgLBD45 Gene downstream vector sequence; c: containing PgLBD45 Partial vector sequence of the gene; d: PgLBD45 gene upstream vector sequence).

[0020] Figure 9 :Overexpression in hairy roots PgLBD45 Relative gene expression.

[0021] Figure 10 : PgLBD45 Detection of the contents of 9 monomer saponins in overexpression-positive hairy roots (Re, Rf, Rg1, Rg2, and Rh1 are protopanaxantriol saponins; Rb1, Rb2, Rg3, and Rh2 are protopanaxadiol saponins. The two types of saponins are distinguished by different colors. "*" represents p ≤ 0.05, “**” represents p ≤ 0.01, “***” represents p ≤ 0.001). DETAILED DESCRIPTION

[0022] Sources 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.

[0023] The ginseng hairy root material was obtained by infection with Agrobacterium C58C1 strain.

[0024] Main content 1. Identification of genes involved in the regulation of ginsenoside biosynthesis This study was based on the Jilin ginseng transcriptome database in the laboratory. Differential expression analysis, correlation analysis, SNP / InDels mutation association analysis, weighted co-expression network analysis and methyl jasmonate-induced expression analysis were used to obtain gene groups highly related to ginsenosides. Combined with the GO functional annotation results, a gene was finally identified as belonging to LBD The genes of the gene family were selected as the research objects and named PgLBD45 .

[0025] 2. Ginseng total RNA extraction and reverse transcription RNA was extracted from ginseng adventitious roots using the Beijing Quanshijin TransZol conventional RNA extraction kit, and the extracted RNA was subjected to agarose gel electrophoresis (Figure 1). First-strand cDNA was synthesized using the Shandong SPARKscript II RT Plus Kit (With gDNA Eraser) reverse transcription kit, and the product was analyzed by agarose gel electrophoresis (Figure 2).

[0026] 3. PgLBD45 Full-length gene ORF cloning Table 1. PgLBD45 Gene length information

[0027] 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 as the first strand template, PgLBD45 PCR amplification was performed, and the PCR product was verified by agarose gel electrophoresis (Figure 3). It was purified using the SanPrep column-based PCR product purification kit of Sangon Biotechnology. TM The 18-T cloning vector was used to transform Escherichia coli.

[0028] 4. Construction of overexpression vector The overexpression vector used in this study was pCAMBIA1300-35S-sGFP (stocked in the laboratory).

[0029] ① Use of pCAMBIA1300-35S-sGFP vector Sac I and Xba I performs linearization double enzyme digestion; ②Use the primer pair PgLBD45-pMD containing homology arms TM 18-T recombinant vector was amplified by PCR to obtain the homology arms PgLBD45 Gene; ③Use the Beijing Quanshijin Basic Seamless Cloning and Assembly Kit to perform homologous recombination on the linearized vector and homology arm gene fragments; ④ Transform the recombinant product into E. coli DH5α competent cells and culture overnight on LB solid medium containing Kan resistance (vector confirmed); ⑤ Pick a single colony and expand the culture in 1 ml of liquid LB medium containing kanamycin (50µg / ml); ⑥ Perform PCR amplification verification on the bacterial solution (Figure 4). Once verified, send it to Sangon Biotech for sequencing verification.

[0030] 5. Preparation of Agrobacterium C58C1 engineered bacteria ①In a clean bench, pipette 5 µl of recombinant plasmid into competent cells, mix well, and place on ice for 5 min; ② Prepare liquid nitrogen in advance, plunge the centrifuge tube into liquid nitrogen for quick freezing for 5 minutes, and then place it in a metal bath (37°C) for heat shock for 5 minutes; ③ In a clean bench, add 700 µl of liquid LB medium (without antibiotics) to the centrifuge tube and culture at 28°C and 170 rpm for 4 h. ④Take out the centrifuge tube and centrifuge at 4000 rpm for 3 minutes ⑤ After discarding the supernatant, resuspend the cells in LB liquid medium (about 100-200µl), spread the cells on LB plates containing rifampicin and kanamycin (determined by the carrier), and incubate them in the dark at 28°C for 48 hours. ⑥ Pick a single colony from the plate, inoculate it into 1 ml of LB liquid medium containing rifampicin and kanamycin (determined by the vector), culture it overnight, and perform PCR verification of the bacterial solution (Figure 5).

[0031] 6. Agrobacterium-mediated transformation of ginseng adventitious roots ① Pre-cultivation Inoculate well-growing ginseng adventitious root segments into MS solid-based culture medium containing 500 µl of hormone 1 and 50 µl of hormone 2 and culture at 23°C in the dark for 2 days.

[0032] ② Co-culture The Agrobacterium engineering bacteria containing the recombinant overexpression vector were amplified to OD 600The bacterial cells were collected by centrifugation and resuspended in 1 / 2 MS liquid medium containing acetosyringone. The bacterial liquid was placed at 28°C and 50 rpm for activation for 1 h. The pre-cultured adventitious root segments were placed in the bacterial liquid and inoculated at 28°C and 80 rpm for 15 min. The bacterial liquid was then aspirated and the root segments were placed in 1 / 2 MS solid medium containing acetosyringone at 23°C and incubated in the dark for 48 h.

[0033] ③ Sterilization culture Place the co-cultivated adventitious roots on filter paper to absorb the bacterial suspension. The roots are then inoculated onto 1 / 2 MS solid medium containing cephalosporin and incubated at 23°C in the dark until hairy roots develop. Only if the hairy roots are still viable and can be propagated on 1 / 2 MS solid medium without hormones can they be used for subsequent positive verification (Figure 6).

[0034] ④ Expansion and cultivation The hairy roots that could be stably subcultured on 1 / 2 MS solid medium were quantitatively transferred into 1 / 2 MS liquid medium for propagation culture. The culture conditions were darkness, 22°C, and 110 rpm (Figure 7).

[0035] 7. Identification of positive hairy roots The genomic DNA of the above-mentioned hairy roots with hormone autotrophic properties 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 the single hairy root as a template, and the amplification results were verified by agarose gel electrophoresis (Figure 8). For single roots that met the expected length, they were PgLBD45 Positive plant material.

[0036] 8. Fluorescence quantitative verification of positive materials Use the method described in point 2 to PgLBD45 RNA was extracted from positive hairy roots and positive hairy roots that only transferred pCAMBIA1300-35S-sGFP empty vector, and reverse transcription was performed using Kangrun Bio StarScript II First-strand cDNA Synthesis Mix With gDNA Remover Reverse Transcription Kit. PgCYP As an internal reference gene, its expression level was verified using Applied Biosystems™ 7500 real-time fluorescence quantitative PCR (Figure 9). PgLBD45 Single root lines with significant changes in gene expression were identified as PgLBD45 Overexpression-positive hairy roots.

[0037] 9. Extraction of saponins from positive hairy roots Dry the liquid-propagated positive hairy roots, weigh 1 g of dry weight, grind, and place in 300 mL of distilled water. Heat and concentrate until 1 / 3 of the volume remains. Remove from heat and filter to collect the filtrate. Repeat this process three times. Combine the filtrates and add them to a fully activated and equilibrated ODS column.

[0038] ① After all the digestion liquid has passed through the ODS column, add 50 mL of 20% methanol to elute the impurities. Repeat this process three times.

[0039] ② After eluting the impurities, add 50 mL of 100% methanol to elute the saponins. Repeat three times and collect the eluate on a rotary evaporator.

[0040] ③ After all the methanol evaporates, crystals will remain. Use 5 mL of chromatographic methanol to dissolve the crystals.

[0041] ④ Filter the dissolved solution with an organic filter (0.22 µm) and place it in a sample bottle to obtain the Panax ginseng adventitious root saponin extract.

[0042] 10. Positive hair root saponin test High-performance liquid chromatography (HPLC) was performed on nine ginsenoside monomer standard solutions, mixed standard solutions, and extracted saponin sample solutions. The HPLC system was a Waters SE 2695, with a Waters C18 column. The mobile phase consisted of 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°C, and the mobile phase flow rate was 1.0 ml / min. The detection wavelength was 203 nm.

[0043] Table 2. HPLC detection time and mobile phase composition ratio

[0044] Calculation formula:

[0045]

[0046] Finally, the saponin data obtained were compared with the saponin data of each super-line and the saponin data of the positive control single root using Welch's t test (no assumption of homogeneity of variance) for two independent samples. The significance is marked with "*" in the figure, as shown in the figure. Figure 10 shown.

[0047] The results showed that 5 PgLBD45 The contents of Rh1 and Rb2 in the overexpression positive hairy roots decreased significantly, while the content of Rb1 increased significantly. PgLBD45 Genes involved in regulating the synthesis of ginsenosides.

Claims

1. A ginseng for regulating ginsenosides PgLBD45 A gene characterized by: The ginseng PgLBD45 The gene sequence is shown in SEQ ID N0.1, and the OFR sequence is shown in SEQ ID N0.

2.

2. A method for increasing ginseng PgLBD45 A primer pair for a gene, characterized in that: The base sequences of the primer pair are shown in SEQ ID N0.3 and SEQ ID N0.

4.

3. Containing the ginseng as claimed in claim 1 PgLBD45 Gene carrier.

4. Containing the ginseng as claimed in claim 1 PgLBD45 Application of genes in regulating the content of ginsenoside monomers.

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