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Notoginseng pathogenesis-related protein gene PnPRlike and application

A technology related to protein and disease course, applied in the fields of molecular biology and genetic engineering, to achieve the effect of shortening the breeding cycle, reducing the use, and reducing environmental pollution

Active Publication Date: 2018-07-06
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disease problem has become a serious obstacle restricting the development of Panax notoginseng planting industry

Method used

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  • Notoginseng pathogenesis-related protein gene PnPRlike and application
  • Notoginseng pathogenesis-related protein gene PnPRlike and application
  • Notoginseng pathogenesis-related protein gene PnPRlike and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: PnPRlike Full-length cDNA cloning and sequence analysis

[0022] Take three-year-old Panax notoginseng leaves to extract total RNA, grind Panax notoginseng leaves into powder with liquid nitrogen, then transfer to a centrifuge tube, extract total RNA by guanidine isothiocyanate method, and then use reverse transcriptase M-MLV (promega) The first strand of cDNA was synthesized using total RNA as a template. The reaction system and operation process were as follows: take 5 μg of total RNA, add 50 ng oligo (dT), 2 μL dNTP (2.5 mM each), and DEPC water in sequence until the reaction volume is 14.5 μL; After mixing, heat denaturation at 70°C for 5 min, then quickly cool on ice for 5 min, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200 U), 1 μL M-MLV (200 U) in sequence, and mix. Homogenize and centrifuge for a short time, incubate at 42 °C for 1.5 h, take out and heat at 70 °C for 10 min to terminate the reaction. After the first strand of cDNA was synthe...

Embodiment 2

[0025] Example 2: Plant overexpression vector construction

[0026] The insert was extracted using the SanPrep column plasmid DNA mini-extraction kit (Shanghai Shenggong). PnPRlike The E. coli plasmid pGEM-T- PnPRlike And the plasmid of the plant expression vector pCAMBIA2300s, take 1 μL for agarose gel electrophoresis to test the integrity and concentration of the extracted plasmid; use restriction endonuclease Bam HI (TaKaRa) and EcoR I(TaKaRa) was applied to the plasmid pGEM-T- PnPRlike Double-enzyme digestion with pCAMBIA2300s (100 μL system), the reaction system and operation process are: take 20 μL pGEM-T- PnPRlike and pCAMBIA2300s plasmid, followed by adding 10 μL 10×Kbuffer, 4 μL Bam HI, 6 μL EcoRI , 60 μL ddH 2 O, centrifuge for a short time after mixing, and place at 37°C for overnight reaction; spot all the digestion products on agarose gel for electrophoresis, and then analyze PnPRlike The fragment and the large fragment of the pCAMBIA2300s vector were g...

Embodiment 3

[0029]Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0030] The transgenic recipient in this experiment is tobacco. Soak the tobacco seeds in 75% alcohol for 30s, wash them with sterile water and wash them with 0.1% HgCl 2 Soak for 8 minutes, then wash several times with sterile water, sow on 1 / 2 MS medium, culture in dark at 28°C for 6 days, transfer to light incubator (25°C, 16h / d light) after germination, and then monthly Subculture once with 1 / 2 MS medium.

[0031] Take out the pCAMBIA2300s- containing pCAMBIA2300s- PnPRlike The plasmid Agrobacterium LBA4404 strain was inoculated in 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampicin, and cultured at 28°C until the medium was turbid. Pipette 1 mL of turbid bacterial solution onto LB solid medium containing 50 mg / L Km, and incubate at 28°C for 48 h; then scrape off an appropriate amount of Agrobacterium on LB solid medium and inoculate it in acetosy...

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Abstract

The invention discloses notoginseng pathogenesis-related protein gene PnPRlike and application. The nucleotide sequence of the PnPRlike gene is shown in SEQ ID NO: 1, which is an encoding pathogenesis-related protein; the related technical research of functional genomics proves that the PnPRlike gene has the function of improving the resistance of plants to pathogenic fungus, and the antifungal gene PnPRlike is constructed onto a plant expression vector and is converted into tobacco for overexpression, transgenic tobacco plant has extremely high in vitro anti-fungal activity, and experimentalresults show that the over-expression transgenic tobacco for PnPRlike has an obvious inhibiting effect for the growth of Fusarium solani, Botryosphaeria dothidea and Nigrospora oryzae.

Description

technical field [0001] The present invention relates to related technologies in the field of molecular biology and genetic engineering, in particular to a pathogen-related protein-like isoform gene with antifungal activity PnPRlike and applications. Background technique [0002] Plants will be stressed by many biotic and abiotic factors in the process of growth and development, such as drought, cold, UV rays, wounds, pathogens (fungi, bacteria, viruses) infection and so on. Correspondingly, plants have evolved a series of defense mechanisms to resist stress, and the activation and accumulation of pathogenesis-related proteins (PRs) is an important part of plant defense responses (Singh A, Kirubakaran SI, Sakthivel N. Heterologous expression of new antifungal chitinase from wheat. Protein Expr Purif, 2007, 56(1):100). When attacked by pathogens, plants respond to pathogens and external stresses by rapidly changing gene expression, inducing the resynthesis of some special pr...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00A01H6/82
CPCC12N15/8282C07K14/415
Inventor 刘迪秋唐笔锋崔秀明杨晓艳熊吟王承潇
Owner KUNMING UNIV OF SCI & TECH
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