Ginsenoside synthesis-regulated PgJAR1 gene as well as encoding protein and application thereof

A technology of ginsenoside and gene coding, which is applied in the fields of application, genetic engineering, DNA/RNA fragments, etc., can solve the problems of unreported research and achieve the effect of high yield, specificity and practicality

Active Publication Date: 2022-05-17
HUNAN INSTITUTE OF ENGINEERING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the cloning of JAR-related genes in ginseng and their application in the synthesis of ginsenosides.

Method used

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  • Ginsenoside synthesis-regulated PgJAR1 gene as well as encoding protein and application thereof
  • Ginsenoside synthesis-regulated PgJAR1 gene as well as encoding protein and application thereof
  • Ginsenoside synthesis-regulated PgJAR1 gene as well as encoding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Acquisition of the PgJAR1 gene

[0037] 1. Ginseng RNA extraction and reverse transcription to prepare cDNA

[0038] The hairy roots induced by fresh 4-year-old ginseng roots were cultured in 1 / 2MS liquid medium at 120rpm and 25°C for 3 weeks in the dark, then 100μmol / L MeJA was added to the medium, at 25°C, and the dark culture was continued for 24h. The roots of ginseng are placed in a mortar pre-cooled with liquid nitrogen, and quickly ground into fine powder after adding liquid nitrogen. Take 30-40mg into a 1.5mL centrifuge tube, add 1mL TRIzol reagent, 40μL β-mercaptoethanol, mix well and place at room temperature for 5-10min; add 0.2mL chloroform, shake for 15s, and place at room temperature for 10-15min; 4°C, 12000 Centrifuge at ×g for 15 min, collect the upper layer and place it in a 1.5 mL centrifuge tube, discard the precipitate; add 0.4 mL of 3mol / L ammonium acetate (pH5.2) and 0.6 mL of isopropanol, mix well, and place at room temperature for 5-10 min, 4 C...

Embodiment 2

[0048] Fluorescent quantitative PCR (qRT-PCR) analysis of the expression levels of PgJAR1 and ginsenoside biosynthetic enzyme genes

[0049] 1. RNA extraction and reverse transcription

[0050] The RNA of hairy roots induced by 4-year-old fresh ginseng roots was extracted, and the hairy roots were cultured in 1 / 2MS liquid medium at 25°C and 110r / min in the dark for 21d, and then added MeJA (100μmol / L) for treatment. Take out the hairy roots for RNA extraction, and the extraction method is the same as in Example 1. The above RNA uses oligod (T)18 as a primer to synthesize cDNA with a pseudo-transcriptase. Primers for qRT-PCR analysis of β-actin, PgJAR1, PgSS, PgSE, PgDDS and PgUGT71A27 genes are as follows:

[0051] β-actin fluorescence quantitative primer F: 5'-TGCCCCAGAAGAGCACCCTGT-3'; (SEQ ID NO.5)

[0052] β-actin fluorescence quantitative primer R: 5'-AGCATACAGGGAAAGATCGGCTTGA-3'; (SEQ ID NO.6)

[0053] PgJAR1 fluorescent quantitative primer F: 5'-GCGGACCTACCGCTATTGAG-...

Embodiment 3

[0068] Extraction and content determination of JA-Ile in ginseng cells

[0069] 1. Extraction of Ginseng JA-Ile

[0070] Take fresh ginseng callus or hair root and grind it into dry powder in liquid nitrogen, weigh an appropriate amount, add isopropanol-water-hydrochloric acid mixed extract, add 8 μL 1 μg / mL internal standard solution, shake at 4 °C for 30 min; add Dichloromethane, shake at low temperature for 30min; centrifuge at 13000r / min at 4°C for 5min, remove the lower organic phase; dry the organic phase with nitrogen in the dark, redissolve with methanol (0.1% formic acid), centrifuge at 13000×g at 4°C for 10min, take The supernatant was passed through a 0.22 μm filter membrane, and JA-Ile was detected by HPLC-MS / MS.

[0071] 2. Determination of ginseng JA-Ile content

[0072] Liquid phase measurement conditions are: Agilent 1290 chromatograph, chromatographic column is Poroshell 120SB-C18 reverse phase chromatographic column (2.1×150, 2.7 μm); Column temperature: 30...

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Abstract

The invention provides a PgJAR1 gene for adjusting ginsenoside synthesis as well as an encoded protein and application of the PgJAR1 gene. The sequence of the PgJAR1 gene for adjusting ginsenoside synthesis is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein of the PgJAR1 gene for adjusting ginsenoside synthesis is shown as SEQ ID NO.2. The invention further provides a preparation method of the PgJAR1 gene for adjusting ginsenoside synthesis. The protein coded by the PgJAR1 gene has an I-type GH3 protein family conserved sequence, belongs to a GH3 gene family in an auxin enzymatic reaction gene, and is jasmonic acid synthase (JAR). The constructed PgJAR1 gene overexpression vector is subjected to agrobacterium-mediated transformation of ginseng callus to obtain PgJAR1 gene overexpressed ginseng cells, the PgJAR1 gene is utilized to regulate the content of endogenous jasmonic acid-isoleucine (JA-Ile) in the ginseng cells so as to regulate the expression of multiple enzyme genes in ginsenoside biosynthesis, the content of JA-Ile is remarkably increased, and the content of JA-Ile is remarkably increased. Further, synthesis and accumulation of ginsenoside are efficiently promoted. The PgJAR1 gene has an important application value in the aspects of increasing the yield of ginsenoside and improving the quality of the ginseng by utilizing the PgJAR1 gene in the ginseng.

Description

technical field [0001] The invention relates to the technical field of biogenetic engineering, in particular to a PgJAR1 gene regulated by ginsenoside synthesis, its encoded protein and its application. Background technique [0002] Ginseng (Panax ginseng C.A. Meyer) is a precious resource of traditional Chinese medicine in my country and one of the most valuable traditional medicinal materials in Asian countries for thousands of years. It is highly praised by Chinese and Western medicine for its extremely high medicinal value. Ginsenosides are the most important medicinal ingredients in ginseng. So far, more than 50 species of ginsenosides have been isolated and identified, including ginsenosides Ra1, Ra2, Ra3, Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rf1, Rg1, Rg2, Rg3, Rh1, Rh2, Rh3, Rs1, Rs2, Ro, CK and CM etc. Modern pharmacological research has found that many ginsenoside monomers have very unique pharmacological effects in anti-tumor, anti-aging, enhancing immunity, anti-mutat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/82A01H5/00A01H6/00
CPCC12N9/93C12Y603/02C12N15/8243
Inventor 张儒谭时泉李昭影张变玲
Owner HUNAN INSTITUTE OF ENGINEERING
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