Gene promoter of ginseng PgPDR3 responded by methyl jasmonate and application thereof
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A technology of methyl jasmonate and promoter, applied in the field of plant genetic engineering
Inactive Publication Date: 2013-05-15
CENT SOUTH UNIV
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However, before the inventor's research, there were no reports on the Panax ginseng PDR protein gene and its regulatory elements, and there was no report on the inducible promoter related to the regulation of the gene expression that affects the synthesis and accumulation of secondary metabolites
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Embodiment 1
[0017] Implementation Example 1 The acquisition of ProPDR3 promoter
[0018] 1. DNA extraction from ginseng root and construction of its promoter library
[0019] 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).
[0020] 2. Primer design and PCR amplification
[0021] (1) Primer design and synthesis
[0022] According to cloned ginseng PgPDR The gene sequence at the 5′ end of the 3 gene (accession number is KC013238, patent application number is 201210462401.X), and the outer and ne...
Embodiment 2
[0033] Construction and transformation of implementation example 2 plant expression vectors
[0034] 1. Construction of plant expression vectors and screening of transgenic tobacco
[0035] (1) According to the promoter sequence design fragments corresponding to the inclusion Bam HI / Nco The specific primers for I restriction sites, the upstream and downstream primers are named P1 and P2 respectively.
[0036] P1: 5'-CGATGGATCCCACTTTGGTTGCTTTCCAATA-3'; (SEQ ID NO. 4)
[0037] P2: 5'-GGGCCATGGTCTTGCTTTCTATACATCACC-3'. (SEQ ID NO.5)
[0038] Use separately Bam HI and Nco I double digested pCAMBIA1301 and ProPDR3, and recovered the pCAMBIA1301 vector and ProPDR3 promoter fragment. The two fragments were ligated and transformed into DH5α, selected by kanamycin, PCR and Bam HI / Nco I double-enzyme digestion identification obtained the recombinant, and the recombinant plasmid was named pCAMBIA1301-ProPDR3::GUS.
[0039] (2) Preparation of Agrobacterium tumefaci...
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Abstract
The invention discloses a gene promoter of ginseng PgPDR3 responded by methyl jasmonate and an application thereof. The sequences of the gene promoter of the ginseng PgPDR3 are shown in SEQ ID NO.1, or are the DNA sequences of which the homology with SEQ ID NO.1 is over 99%. According to the invention, a Genomewalker technology is adopted to clone, later the sequences of the gene promoter of the PDR transport protein is obtained, and then the expression vector Pcambia1301-ProPDR3::GUS of the promoter is built; if the promoter in the transgenic tobacco drives the GUS to express, and can be controlled by ginsenoside, salicylic acid, gibberellins and abscisic acid, the promoter participates in the transport and accumulation of the ginsenoside, which provides a basis for cloning the gene of key control recording factor in the ginsenoside transport signal pathway and controlling the transport and accumulation of the terpenoid, such as ginsenoside; and fine plants containing rich ginsenoside can be obtained by using a method for controlling or improving the ginseng and other plants via the promoter or the derivative thereof.
Description
technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a promoter of a ginseng PDR transporter gene which responds to methyl jasmonate. 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 synthesis and a...
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