Ginseng PgJAZ1 gene, method for improving protopanaxatriol saponin based on gene and application

A ginseng and gene technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of JAZ gene cloning and functional research that have not been reported

Active Publication Date: 2021-10-26
HUNAN INSTITUTE OF ENGINEERING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There is no report about the cloning and functional study of JAZ gene in ginseng

Method used

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  • Ginseng PgJAZ1 gene, method for improving protopanaxatriol saponin based on gene and application
  • Ginseng PgJAZ1 gene, method for improving protopanaxatriol saponin based on gene and application
  • Ginseng PgJAZ1 gene, method for improving protopanaxatriol saponin based on gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Acquisition of the PgJAZ1 gene

[0031] 1. Ginseng RNA extraction and reverse transcription

[0032] After culturing the hairy roots of ginseng at 25°C for 3 weeks, add 100 μmol / L MeJA to the culture medium, culture at 120 rpm, 25°C for 24 hours in the dark, extract total RNA, and use oligod(T) 18 As a primer, the first strand of cDNA was synthesized by reverse transcription with reverse transcriptase, and the synthesized cDNA was stored at -20°C for future use.

[0033] 2. PgJAZ1 gene amplification

[0034] According to the MeJA-induced ginseng transcriptome sequencing results, primers were designed for PCR amplification. PgJAZ1 gene PCR amplification primers are as follows.

[0035] PgJAZ1-F: 5'-ATGTTTCTTCAACGGCCGGAAAT-3'; (SEQ ID NO.4)

[0036] PgJAZ1-R: 5'-CTATAAGTTGAGATCAAACTG-3'; (SEQ ID N0.5)

[0037] The PCR amplification conditions were: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 50°C for 20 s, extension at 72°C for 30 ...

Embodiment 2

[0039] Relative quantitative analysis of PgJAZ1 gene expression

[0040] 1. RNA extraction and reverse transcription

[0041] The hairy roots of ginseng were cultured in 1 / 2MS liquid medium at 25°C and 110r / min in the dark for 21 days, and were treated with MeJA (100 μmol / L) respectively. T) 18 As a primer, the first strand of cDNA was synthesized by reverse transcription with reverse transcriptase. Primers for qRT-PCR analysis of PgJAZ1, β-actin, PgSS, PgSE and PgDDS genes are as follows:

[0042] PgJAZ1 fluorescent quantitative primer F: 5'-GAGAGACCTGCTGCAATGGA-3'; (SEQ ID NO.6)

[0043] PgJAZ1 fluorescent quantitative primer R: 5'-GGGGGCATGTTGAGGAAAGA-3'; (SEQ ID NO.7)

[0044] β-actin fluorescence quantitative primer F: 5'-TGCCCCAGAAGAGCACCCTGT-3'; (SEQ ID NO.8)

[0045] β-actin fluorescent quantitative primer R: 5'-AGCATACAGGGAAAGATCGGCTTGA-3'; (SEQ ID N0.9)

[0046] PgSS fluorescent quantitative primer F: 5'-ATCCCTCCGGAGCCACACTGG-3'; (SEQ ID NO.10)

[0047] PgSS f...

Embodiment 3

[0055] Ginsenoside Extraction and Content Determination

[0056] 1. Extraction of ginsenosides

[0057] Take fresh ginseng hair roots or tissues, wash them with tap water for 2 minutes, and then use ddH 2 O washed, dried at 60°C to constant weight. Grind it into a fine powder, leaching with 80% methanol at 60°C (1g:40mL), ultrasonic treatment 3 times, 15min each time; Saturated n-butanol extraction, and the n-butanol layer was collected. Evaporate n-butanol to dryness in a water bath at 60°C to obtain total ginsenosides, dissolve with an appropriate amount of methanol ultrasonically, set the volume to the mark, and pass through a 0.45 μm microporous membrane to obtain a sample solution.

[0058] 2. Determination of ginsenoside content

[0059] Ginsenoside content determination adopts HPLC method, and HPLC measuring condition is: LC-MS 8050 high performance liquid chromatography; Chromatographic column is ACQUITY UPLC BEH Shield RP18 column (1.7 μ m, 2.1mm * 50mm); Mobile p...

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Abstract

The invention discloses a ginseng PgJAZ1 gene, a method for improving protopanaxatriol saponin based on the gene and application of the ginseng PgJAZ1 gene. The gene is derived from Panax ginseng C.A.Meyer and is named as PgJAZ1, and a protein coded by the gene has a conservative TIFY structural domain and a jas structural domain which are specific to the JAZ family. The constructed PgJAZ1 gene RNA interference vector is used for transforming a ginseng root, and a PgJAZ1 gene silenced ginseng hairy root is obtained. Compared with a control ginseng hairy root, the content of protopanaxatriol saponin and the content of total saponin in the PgJAZ1 gene silenced ginseng hairy root are remarkably increased. Researches show that the PgJAZ1 gene is utilized to regulate the content of endogenous jasmonic acid methyl ester in ginseng hairy roots so as to regulate the biosynthesis of protopanaxatriol type saponins and total saponins. The invention has important application value in the aspects of increasing the yield of the protopanaxatriol saponin and improving the quality of the ginseng by utilizing the PgJAZ1 gene in the ginseng.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a ginseng PgJAZ1 gene and a method and application for increasing protopanaxatriol-type saponins based on the gene. Background technique [0002] Ginsenosides are the most important medicinal ingredients in the precious traditional Chinese medicine ginseng in my country. Ginsenosides are divided into dammarane-type and oleanolic acid-type saponins according to the position, type and quantity of glycosidic bonds on the mother nucleus. Marane-type saponins are divided into Protopanaxdiol-type saponins (Protopanaxdiol-type, PPD type, mainly including Rb1, Rb2, Rc and Rd, etc.) and Protopanaxtriol-type saponins (Protopanaxtriol-type, PPT type, including Re and Rg1 etc). So far, more than 100 ginsenosides have been isolated and their structures determined (J Ginseng Res 2020; 44(4):552–62), and PPT-type ginsenosides account for about 30% of the total saponins. The Chine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N15/113A01H5/06A01H6/00
CPCC07K14/415C12N15/8218C12N15/8243C12N15/8205
Inventor 张儒谭时泉张变玲
Owner HUNAN INSTITUTE OF ENGINEERING
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