A ginseng PgGUAT252645 gene and its application

Abstract

The present invention first discovered a uridine diphosphate glucuronyl transferase gene, PgGUAT252645, directly involved in the biosynthesis of ginsenoside Ro in ginseng, and the gene sequence is shown in SEQ ID No. 1. The gene is the mRNA of the uridine diphosphate glucuronyl transferase that specifically glycosylates and modifies the C3 position of oleanolic acid in the synthesis process of ginsenoside Ro in ginseng. The SNP mutation that can cause amino acid substitution at 622bp of its open reading frame can significantly affect the content of ginsenoside Ro. The unmutated PgGUAT252645 gene can increase the content of ginsenoside Ro, and the mutated PgGUAT252645 gene can reduce the content of ginsenoside Ro. This discovery is of great significance to ginseng breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a ginsenoside Ro synthesis-related gene PgGUAT252645 and an application thereof. Background Art

[0002] Ginseng (Panax ginseng CAMeyer), a perennial herbaceous plant of the genus Panax in the Araliaceae family, is a traditional precious Chinese herbal medicine. Due to its great medicinal value, it is widely used in the clinical treatment of various diseases and the production of health food and other commodities worldwide. Ginsenosides are secondary metabolites of Panax plants and are also the main pharmacological components of ginseng. The different types of triterpenoid molecules, glycosylation positions, glycosyl types and glycosyl numbers in the molecular structures of various ginsenosides determine their diverse pharmacological activities.

[0003] Ginsenoside Ro is the only reported oleanane-type pentacyclic triterpene ginsenoside derived from ginseng. Its known pharmacological activities include inhibiting tumor growth, anti-thrombosis, anti-inflammation, and improving obesity. In addition, studies have pointed out that, just as ginsenosides Rb1 and Re can participate in regulating the growth of ginseng adventitious roots, ginsenoside Ro has the effect of promoting the growth of ginseng adventitious roots at low concentrations and inhibiting it at high concentrations. The synthesis pathway of ginsenoside Ro is exactly the same as that of tetracyclic triterpene ginsenosides before the precursor beta-amyrin. Both use 2,3-oxidized squalene generated by the final product IPP of the MVA and MEP pathways as a substrate for the synthesis of the aglycone skeleton. Ginsenoside Ro is a glycosylated product of oleanolic acid generated by beta-amyrin catalyzed by oleanolic acid synthase. These glycosylation include modification of the glucuronic acid group at the C3 position of oleanolic acid, modification of the glucose group at the C2 position of the glucuronic acid group, and modification of the glucose group at the C28 position of notoginseng ginger saponin R1 or panax notoginseng saponin IVa.

[0004] However, there are still few studies on genes related to the synthesis of ginsenoside Ro. Currently, only the cloning and characterization of the synthase genes of precursors such as β-amyrin and oleanolic acid, as well as a group of glycosyltransferases belonging to different gene families from ginseng, ginger-shaped Panax notoginseng, Panax notoginseng and Panax japonicus, have been confirmed to be involved in the glycosylation modification step in the synthesis of ginsenoside Ro. However, due to the complexity of the metabolic network, many key genes and regulatory factors related to this synthesis pathway have not yet been revealed. Therefore, continuing to deeply explore the genes related to the synthesis of ginsenoside Ro is of great significance to enrich the ginseng gene resources and further reveal the mechanism of ginsenoside synthesis. Summary of the invention

[0005] The purpose of the present invention is to provide a ginseng PgGUAT252645 gene.

[0006] Another object of the present invention is to provide an application of ginseng PgGUAT252645 gene in the synthesis of ginsenoside Ro.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A ginsenoside Ro synthesis-related gene PgGUAT252645, the open reading frame base sequence of the gene is shown as SEQ ID No.1.

[0009] A ginseng PgGUAT252645 gene is used in the synthesis of ginsenoside Ro, and the specific method is as follows:

[0010] PgGUAT252645 is the mRNA of uridine diphosphate glucuronyl transferase that specifically glycosylates and modifies the C3 position of oleanolic acid in the synthesis of ginsenoside Ro in ginseng; SNP mutations that can cause amino acid substitutions at 622bp in its open reading frame can significantly affect the content of ginsenoside Ro, such as Figure 1 shown.

[0011] The present invention has the following beneficial effects:

[0012] For the glycosylation modification step in the synthesis pathway of ginsenosides in ginseng, there is not only the phenomenon of mixed catalysis that a certain glycosyltransferase can catalyze the glycosylation modification of multiple substrates, but also the phenomenon that multiple glycosyltransferase isozymes co-catalyze the glycosylation modification of a substrate, and these glycosyltransferase isozymes may come from different gene families. Similar to the study of substrate preference, the main pathways in the metabolic network of glycosylation modification of ginsenosides accompanied by mixed catalysis can be clarified. The exploration of the primary and secondary or complementary relationships between each isozyme and the in-depth mining of other isozyme members will further analyze the synthesis process of ginsenosides.

[0013] The present invention is the first to discover in ginseng a uridine diphosphate glucuronyl transferase gene, PgGUAT252645, which is directly involved in the biosynthesis of ginsenoside Ro, and its open reading frame base sequence is shown in SEQ ID No. 1. The gene is the mRNA of the uridine diphosphate glucuronyl transferase that specifically glycosylates and modifies the C3 position of oleanolic acid in the synthesis process of ginsenoside Ro in ginseng. The SNP mutation that can cause amino acid substitution at 622bp of its open reading frame can significantly affect the content of ginsenoside Ro. The unmutated PgGUAT252645 gene can increase the content of ginsenoside Ro, and the mutated PgGUAT252645 gene can reduce the content of ginsenoside Ro. This discovery is of great significance to ginseng breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1:Effect of SNP mutation at 622bp of PgUGAT252645 open reading frame on ginsenoside Ro.

[0015] Figure 2 :SDS-PAGE electrophoresis to detect the induced expression of the protein (lanes 1 and 4 are the supernatant samples of cell lysate without induced expression, lanes 2 and 5 are the supernatant samples of cell lysate induced by IPTG, and lane 3 is the protein molecular weight marker; the * marks the position of the specific expression protein band).

[0016] Figure 3 : Liquid chromatography-mass spectrometry detection of catalytic reaction system extracts (A. Liquid chromatography analysis of standard and extract samples. The red curve is the standard solution containing oleanolic acid (OA) and calendula saponin E (CE), and the green and blue curves represent the extract samples of different catalytic reaction systems; B. Standard mass spectrometry detection results).

[0017] Figure 4 :Induction and culture of ginseng hairy roots (A. Ginseng adventitious roots; B. Pre-culture; C. Sterilization culture after infection; D. Development of ginseng hairy roots; E. Proliferation and subculture of ginseng hairy roots; F. Liquid culture of ginseng hairy roots).

[0018] Figure 5 : Expression level of target gene and content of ginsenoside Ro in transgenic ginseng hairy roots (blue columns in A and B represent the content of ginsenoside Ro and the relative expression level of PgUGAT252645ORF in samples; orange columns in C and D represent the content of ginsenoside Ro and the relative expression level of PgUGAT252645ORF622M in samples; gray columns represent the content of ginsenoside Ro and the expression level of internal reference gene in the control group, respectively). DETAILED DESCRIPTION

[0019] Example 1 Prokaryotic expression of PgGUAT252645 gene and detection of catalytic activity

[0020] The open reading frame sequence of PgGUAT252645 (PgUGAT252645ORF) is shown in SEQ ID No. 1. The sequence and the mutant sequence at 622 bp (PgUGAT252645ORF622M) were artificially synthesized and ligated to the restriction sites BamH I and Hind III of the plasmid vector pET-28a to construct a prokaryotic expression plasmid. The constructed recombinant plasmids were transformed into chemically competent cells of Escherichia coli BL21 (DE3) to construct a recombinant expression strain. The positive strains obtained by screening were cultured at 37°C and 180 rpm until OD600 reached 0.8, and then isopropyl-beta-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM was added, and protein expression was continuously induced for 12 hours at 16°C and 180 rpm. Then, the bacterial cells were collected by centrifugation at 4°C and 12,000 rpm for 15 minutes, resuspended in HEPES buffer, and then broken by ultrasonic cell disruptor. Finally, the supernatant was collected by centrifugation at 4°C and 12,000 rpm for 45 minutes for SDS-PAGE protein electrophoresis detection and catalytic activity detection.

[0021] The catalytic reaction system with a volume of 100 μL includes 85 μL of the above supernatant, 0.5 mM oleanolic acid as the acceptor substrate, and 5 mM uridine-5'-diphosphoglucuronic acid (UDPGA) as the glycosyl donor substrate. After incubation at 30°C for 12 h, an equal volume of water-saturated n-butanol was added to terminate the reaction, and then the organic phase was separated and evaporated to dryness. The residue was dissolved in chromatographic methanol and analyzed by liquid chromatography-mass spectrometry under the detection conditions shown in Table 1.

[0022] The results of SDS-PAGE protein electrophoresis were as follows: Figure 2 As shown in the figure, compared with the control group without adding IPTG for induction expression, specific protein bands appeared in the supernatant lane of cell disruption in the experimental group, indicating successful induction of expression. Figure 3 As shown, in the reaction system containing PgUGAT252645ORF protein, the glucuronidation modification product of oleanolic acid C3, calendula saponin E, was detected, while in the reaction system containing PgUGAT252645ORF622M protein, no corresponding glucuronidation modification product was detected.

[0023] Table 1. HPLC-MS / MS analysis conditions for catalytic activity of prokaryotic expression products

[0024]

[0025] Example 2 Induction and culture of ginseng hairy roots overexpressing PgUGAT252645ORF and PgUGAT252645ORF622M

[0026] PgUGAT252645ORF and PgUGAT252645ORF622M were artificially synthesized and ligated to the restriction sites Sac I and Sal I of the plasmid vector pCAMBIA1300-35S to construct an overexpression plasmid. The hairy root samples induced by Agrobacterium C58C1 carrying the blank plasmid pCAMBIA1300-35S were used as controls. The constructed recombinant plasmids were transformed into Agrobacterium C58C1 competent cells to construct recombinant strains for infection. The positive strains obtained by screening were cultured at 28°C and 170rpm until OD600 reached 0.4, and then centrifuged at 4°C and 12000rpm for 15min to collect bacterial cells, and resuspended with 1 / 2MS liquid culture medium containing acetosyringone (AS), and the cell concentration was adjusted to OD600=0.5. Then the pre-cultured adventitious roots of ginseng were added to the bacterial solution for infection. The infected adventitious roots were first cultured on 1 / 2MS solid medium containing 20μM AS for 48h, and then transferred to 1 / 2MS solid medium containing cephalosporin for further culture until hairy roots developed. The asexual single root system of hairy roots with stable growth during the subculture process was selected and expanded in liquid 1 / 2MS medium at 22℃ and 110rpm. The induction and culture process was as follows Figure 4 shown.

[0027] Example 3 Detection of the expression level of target gene and the content of ginsenoside Ro in transgenic ginseng hairy roots

[0028] The dried and crushed samples of each transgenic hairy root asexual single root system were used for ginsenoside extraction. Three biological replicates were set for each single root system, and the ginsenosides in the water decoction of each sample were extracted using the ODS method, and then analyzed by liquid chromatography-mass spectrometry under the detection conditions shown in Table 2.

[0029] The cryopreserved samples of each transgenic hairy root asexual single root system were used for total RNA extraction. Three biological replicates were set up for each single root system. The total RNA was extracted using the TRIpure total RNA extraction kit, and then the genomic DNA was removed and reverse transcribed into cDNA using the HiFiScript rapid genome-free cDNA first-strand synthesis kit. Subsequently, cDNA was used as a template and UltraSYBR Mixture (Low ROX) was used to detect the expression level of PgUGAT252645ORF or PgUGAT252645ORF622M in each sample by real-time fluorescence quantitative PCR. Three technical replicates were set up for each sample, and the actin gene Actin1 was selected as the internal reference gene. After the expression level of the internal reference gene was normalized, 2-ΔΔCt The target gene expression was calculated.

[0030] The detection results of ginsenoside Ro content and PgUGAT252645ORF or PgUGAT252645ORF622M expression in each sample are as follows: Figure 5 Compared with the hairy root samples induced by Agrobacterium C58C1 carrying the blank plasmid pCAMBIA1300-35S as the control, the expression levels of PgUGAT252645ORF in the five PgUGAT252645ORF-overexpressing hairy root asexual monoroot systems increased to varying degrees, ranging from 1.509-3.967 times ( Figure 5 A). At the same time, the content of ginsenoside Ro in 4 samples increased significantly, and was correlated with the expression increment ( Figure 5 B). Although the content of ginsenoside Ro in some samples of asexual single root systems with overexpression of PgUGAT252645ORF622M was also increased, the increase was generally much smaller than that in samples with overexpression of PgUGAT252645ORF and was not related to the expression increment ( Figure 5 C, D).

[0031] Table 2. HPLC-MS / MS analysis conditions for ginsenoside Ro content in ginseng hairy roots

[0032]

Claims

1. An application of the ginseng PgGUAT252645 gene in the synthesis of ginsenoside Ro, characterized in that: The open reading frame base sequence of the ginseng PgGUAT252645 gene is shown in SEQ ID No.

1. The PgGUAT252645 gene is the coding gene for the mRNA of the uridine diphosphate glucuronyl transferase that specifically glycosylates and modifies the C3 position of oleanolic acid in the synthesis of ginsenoside Ro.

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