PDR transport protein gene promoter for controlling ginsenoside accumulation, and its application

A technology of ginsenoside and transporter, applied in the field of plant genetic engineering

Inactive Publication Date: 2013-05-08
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports of PDR protein genes and their regulatory elements in ginseng

Method used

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  • PDR transport protein gene promoter for controlling ginsenoside accumulation, and its application
  • PDR transport protein gene promoter for controlling ginsenoside accumulation, and its application
  • PDR transport protein gene promoter for controlling ginsenoside accumulation, and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1 Obtaining of ProPDR1 promoter

[0021] 1. DNA extraction from ginseng root and construction of its promoter library

[0022] Four-year-old ginseng roots from Jilin Changbai Mountain ginseng production area were taken, soaked in tap water for 45 minutes, rinsed twice, first soaked in 75% ethanol for 30 seconds (sec), and rinsed twice with sterile water. Refer to the instructions of Omega Plant DNA Kit (Omega Company) to extract genomic DNA, use the total DNA of ginseng root as a template, and construct according to the instructions of Genomewalker Kit (Takara Company) Dra I library (DL1), Eco R V library (DL2), Pvu II library (DL3) and Stu I library (DL4).

[0023] 2. Primer design and PCR amplification

[0024] (1) Primer design and synthesis

[0025] According to cloned ginseng PgPDR 1 Gene sequence at the 5′ end of the gene (accession number KC013236, patent application number 201210464442.2), design outer and nested primers, and use Primer Pre...

Embodiment 2

[0036] Example 2 Construction and Transformation of Plant Expression Vectors

[0037] 1. Construction of plant expression vectors

[0038] (1) According to the ProPDR1 promoter sequence design fragments corresponding to the inclusion Hin d III / Bam The specific primers for the HI restriction site, the upstream and downstream primers are named P1 and P2 respectively.

[0039] P1: 5'-CGCGGATCCATCTTATACTCCATCCGTCCC-3'; (SEQ ID NO.4)

[0040] P2: 5'-CCCAAGCTTAATTAACATCAGAATACCCAGA-3'. (SEQ ID NO.5)

[0041] Use separately Hin d III and Bam HI double digestion of pBI121 and ProPDR1, recovery of pBI121 vector and ProPDR1 promoter fragments. The two fragments were ligated and transformed into DH5α, selected by kanamycin, PCR and Hin d III / Bam The recombinant was identified by HI double digestion, and the recombinant plasmid was named pBI121-ProPDR1::GUS.

[0042] (2) Preparation of Agrobacterium tumefaciens competent cells

[0043] ① Streak culture of Agrobacteri...

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Abstract

The invention discloses a PDR transport protein gene promoter for controlling ginsenoside accumulation, and its application. A promoter ProPDR1 has a DNA sequence represented by SEQIDNo.1 or has a DNA sequence having a homology of above 99% to SEQIDNo.1. A PgPDR1 transport protein gene promoter sequence is obtained through cloning by adopting genome walker in the invention, and includes a TCA element and TCCACCT-Motif. A promoter expression vector pBI121-ProPDR1::GUS is constructed in the invention, and reporter gene GUS is strongly controlled by ginsenoside, salicylic acid, methyl jasmonate and abscisic acid in transgenic tobacco under the driving of the promoter ProPDR1, so the ginseng PgPDR1 gene expression promoted by the promoter is closely related to the ginsenoside accumulation. The promoter provided by the invention has very important application values in the medical plant secondary metabolite synthesis and accumulation control gene engineering.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a promoter of a PDR transport protein gene capable of regulating ginsenoside accumulation. Background technique [0002] Ginseng ( P. ginseng C.A. Meyer) is a perennial herb of the Araliaceae ginseng genus, and is a precious Chinese medicinal material. Ginseng contains a variety of medicinal ingredients, among which ginsenoside is the main active ingredient of ginseng. At present, more than 50 kinds of ginsenosides have been isolated from ginseng roots. Modern medical research has proved that each saponin monomer has important medicinal value and has been widely used in clinical practice. However, some of them have very low content of saponins with strong activities such as anti-tumor, anti-aging, inhibiting cell apoptosis and enhancing immunity. Using gene regulation technology related to ginsenoside synthesis and metabolism to promote the synthe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/82C12N15/66A01H5/00
Inventor 罗志勇张儒黄景嘉陈湘晖罗俊李继佳
Owner CENT SOUTH UNIV
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