Pathogenesis-related protein 1 family gene PnPR1-2 of panax notoginseng and application of pathogenesis-related protein 1 family gene PnPR1-2

A technology related to protein and disease course, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of long period of breeding resistant varieties, time-consuming and laborious farming system, and high chemical pesticide residues, so as to shorten the breeding period and save costs. , the effect of broad market application prospects

Active Publication Date: 2016-12-21
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, these methods have more or less disadvantages, such as the long period of breeding resistant varieties, high chemical pesticide residues and easy to cause environ...

Method used

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  • Pathogenesis-related protein 1 family gene PnPR1-2 of panax notoginseng and application of pathogenesis-related protein 1 family gene PnPR1-2
  • Pathogenesis-related protein 1 family gene PnPR1-2 of panax notoginseng and application of pathogenesis-related protein 1 family gene PnPR1-2
  • Pathogenesis-related protein 1 family gene PnPR1-2 of panax notoginseng and application of pathogenesis-related protein 1 family gene PnPR1-2

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Experimental program
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Effect test

Embodiment 1

[0021] Example 1: PnPR1-2 Full-length gene cloning and sequence analysis

[0022] The roots of annual Panax notoginseng were inoculated with Fusarium solani rot, and total RNA was extracted from the roots 12 h after inoculation. Use reverse transcriptase M-MLV (promega) to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process are as follows: Take 5 μg TotalRNA, add 50 ng oligo (dT), 2 μL dNTP (2.5mM each) in sequence , DEPC water to a reaction volume of 14.5 μL; after mixing, heat and denature at 70°C for 5 minutes, then quickly cool on ice for 5 minutes, then add 4 μL 5×First-standbuffer, 0.5 μL RNasin (200U), 1 μL M- MLV (200U), mix well and centrifuge for a short time, incubate at 42°C for 1.5 h, take it out and heat at 70°C for 10 min to terminate the reaction. After the first strand of cDNA was synthesized, it was stored at -20°C for future use.

[0023] Use the synthesized first-strand cDNA as a template to amplif...

Embodiment 2

[0025] Embodiment 2: plant overexpression vector construction

[0026] Use the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert PnPR1-2 coli plasmid pGEM-T- PnPR1-2 As well as the plasmid of the plant expression vector pCAMBIA2300s, take 1 μL for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid; Bam HI (TaKaRa) and Eco RI (TaKaRa) respectively on the plasmid pGEM-T- PnPR1-2 and pCAMBIA2300s for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pGEM-T- PnPR1-2 and pCAMBIA2300s plasmid, respectively add 10 μL 10×Kbuffer, 5 μL BamHI , 5 μL Eco RI, 60 μL ddH 2 O, mix well and then centrifuge for a short time, and place it at 37°C for overnight reaction; spot all the digested products on agarose gel for electrophoresis, and then separate PnPR1-2 The fragments and the large fragments of the pCAMBIA2300s vector were recovered b...

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- PnPR1-2 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 the Agrobacterium on LB solid medium and inoculate it in an appropriate amount of ace...

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Abstract

The invention discloses a pathogenesis-related protein 1 family gene PnPR1-2 of panax notoginseng. The nucleotide sequence of the pathogenesis-related protein 1 family gene PnPR1-2 is as shown in SEQ ID No. 1. The pathogenesis-related protein 1 family gene PnPR1-2 comprises protein encoded with an amino acid sequence as shown in SEQ ID No. 2. Related technological researches of functional genomics prove that the PnPR1-2 gene is capable of increasing the fungus resistance of plants; when the PnPR1-2 gene is built onto a plant expression carrier and transferred into tobacco for overexpression, the transgenic tobacco plant is high in in-vitro antifungal activity, and the overexpression PnPR1-2 transgenic tobacco has an evident inhibiting effect on the growth of various fungi such as Fusarium solani, Verticillium Fusarium, beaded gibberella and Alternaria panax.

Description

technical field [0001] The invention relates to the field of molecular biology and genetic engineering related technology research, in particular to a Panax notoginseng disease course-related protein 1 family gene with antifungal activity PnPR1-2 and applications. Background technique [0002] Plant diseases are a very difficult problem in agricultural production, especially fungal diseases, which account for about 80% of the total plant diseases and seriously affect the yield and quality of crops. Traditional disease control methods have achieved certain results. One is to rely on traditional breeding methods to cultivate resistant varieties, the other is to use chemical pesticides, and the third is to adopt farming systems such as crop rotation. However, these methods have more or less disadvantages, such as the long period of breeding resistant varieties, high chemical pesticide residues and easy to cause environmental pollution, and time-consuming and laborious adjustme...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/84C07K14/415A01H5/00
CPCC07K14/415C12N15/8205C12N15/8282
Inventor 刘迪秋崔秀明白智伟曲媛杨野关瑞攀陈瑞
Owner KUNMING UNIV OF SCI & TECH
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