ABC transporter PgPDR2 gene and encoding protein and application of ABC transporter gene PgPDR2
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A transport protein and gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc.
Inactive Publication Date: 2013-02-13
CENT SOUTH UNIV
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Furthermore, no plant PDR proteins have been reported to transport more complex terpenoid molecules
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Embodiment 1
[0022] The acquisition of embodiment 1 PgPDR2 gene
[0023] 1. Ginseng suspension cell culture
[0024] (1) Take four-year-old ginseng roots from the ginseng production area of Changbai Mountain in Jilin, soak in tap water for 45 minutes, rinse twice, first soak in 75% ethanol for 30 seconds (sec), rinse twice with sterile water, and use it on the ultra-clean workbench in the sterile room Sterilize with 0.1% HgCl2, stir constantly, rinse with sterile distilled water 4-5 times.
[0025] (2) Under sterile conditions, inoculate the sterilized explants into MS medium containing 2,4-D (2.0 mg / L) and KT (0.5 mg / L). After 3 subcultures, the explants were completely dedifferentiated. After the callus was completely formed, 6 explants were inoculated per 100ml Erlenmeyer flask. Subculture to supplement the nutrients and other requirements of the culture. The first subculture was carried out 30 days after inoculation, and then every 3-4 weeks.
[0026] (3) After the callus was obt...
Embodiment 2
[0091] Implementation example 2 construction and transformation of expression vector
[0092] 1. Construction of plant overexpression vectors and screening of transgenic plants
[0093] (1) Digest pBI121 and PgPDR2 with BamHI and Sac I respectively, and recover pBI121 vector and PgPDR2 gene fragment. The two fragments were connected and transformed into DH5α, and the recombinant was screened out, and the recombinant plasmid was named pBI-PgPDR2. The upstream and downstream primers for amplifying the PgPDR2 gene are respectively:
[0094] PgPDR2:F1:5'-CG GGATCCCGCACCATGGATGGAAGTAATCTGTA-3'; (SEQ ID NO. 11)
[0095] R1:5'-CGC GAGCTC GGCTCACCGCCTTTGGAAGTTGA-3'. (SEQ ID NO.12)
[0096] The underlined part is the restriction site of BamH I and Sac I.
[0097] (2) Preparation of Agrobacterium tumefaciens competent cells
[0098] ① Streak culture of Agrobacterium LBA4404 on YEB plate containing 100 μg / ml rifamycin, culture in dark at 28°C for 2 days.
[0099] ②Pick a singl...
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Abstract
The invention discloses a PgPDR2 (panax ginseng pleiotropic drug resistance) gene related to regulation of transport and accumulation of ginsenosides, as well as an encoding protein and an application of the PgPDR2 gene. A sequence of the ABC (ATP-binding cassette) PgPDR2 gene is shown as SEQ ID No. (sequence identification number) 1, and the sequence of the encoding protein of the ABC PgPDR2 gene is shown as SEQ ID No. 2. The research finds that PgPDR2 gene expression has tissue specificity and is related to the accumulation of the ginsenosides. The research preliminarily indicates that the PgPDR2 gene has functions of promoting synthesis, the transport and the accumulation of the ginsenosides. Panax ginseng or other plants can be screened or improved through the PgPDR2 gene or derivatives of the PgPDR2 gene to obtain excellent plant varieties with high ginsenoside content. Also, panax ginseng transgenic cells or plants can be adopted to produce ginsenoside monomers such as protopanaxadiol, Rb, Re, Rh2, Rg2 and Rg3.
Description
technical field [0001] The invention belongs to the technical field of biogenetic engineering, and relates to improving plant traits, in particular to related genes for promoting ginsenoside biosynthesis, translocation and accumulation, proteins and applications thereof. The invention has important application value in improving the content of ginsenoside in ginseng and other plants. Background technique [0002] Ginseng (P.ginseng C.A.Meyer) is a perennial herb of the Araliaceae genus Panax, 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, a...
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