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Panax notoginseng disease resistance related protein pnpr10-2 and its coding gene and application

A related protein, Panax notoginseng technology, applied in application, genetic engineering, plant genetic improvement and other directions

Active Publication Date: 2020-12-15
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the prevention and control of root rot is mainly through the method of large-scale application of chemical pesticides, which has a certain degree of influence on the quality and safety of medicinal materials. However, no Panax notoginseng varieties with good disease resistance potential to root rot have been found so far. Therefore, through cloning Root rot resistant PR gene in Panax notoginseng, research on its disease resistance is of great significance for cultivating and screening disease-resistant varieties, and using molecular breeding methods to cultivate new varieties of root rot-resistant Panax notoginseng and other horticultural crops

Method used

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  • Panax notoginseng disease resistance related protein pnpr10-2 and its coding gene and application
  • Panax notoginseng disease resistance related protein pnpr10-2 and its coding gene and application
  • Panax notoginseng disease resistance related protein pnpr10-2 and its coding gene and application

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1, the acquisition of the PnPR10-2 conserved fragment and its full-length cDNA

[0024] The total RNA from the roots of Panax notoginseng was extracted using the Shanghai Sangong Column Plant Total RNA Extraction and Purification Kit (SK8662), and RNA electrophoresis was performed for verification after extraction. After successful RNA extraction, reverse transcription reaction was performed using TaKaRa PrimeScript RT reagent Kit for subsequent experiments.

[0025] According to the sequence of the Panax notoginseng PR10-2 gene predicted in the transcriptome cDNA database, a full-length specific primer PR10-2-F containing a restriction site was designed (5'-GGATCCatgggtgtccaaaagaccgaaac-3', GGATCC is the BamHI restriction site) and PR10-2-R (5'-GAATTCctaatttgctaggaggtaagcttcaacagc-3', GAATTC is the EcoRI restriction site).

[0026] The cDNA of the main root of biennial Panax notoginseng was used for amplification. The PCR reaction conditions were: 94°C for 3mi...

Embodiment 2

[0031] Embodiment 2, construction of PnPR10-2 prokaryotic expression vector, expression and purification of recombinant protein

[0032] 1. Construction of pET32a-PnPR10-2 prokaryotic expression vector

[0033] (1) Plasmid extraction: pick out the single clone colony containing the pET-32a empty vector and the positive clone colony of pMD-18T-PnPR10-2 with correct sequencing, and extract the plasmid after shaking;

[0034] (2) Enzyme digestion and gel recovery: The correct pMD-18T-PnPR10-2 carrier plasmid detected by sequencing was double-digested with restriction endonucleases BamHI and EcoRI, detected by agarose gel electrophoresis and recovered with a DNA recovery kit;

[0035] (3) Ligation: Ligate the recovered target fragment with the digested pET32a empty vector with DNA ligation kit, transform Trans1T1 competent cells, spread on Amp plate, pick three single colonies after 12 hours, perform PCR detection and send to Shanghai Biosequencing verification;

[0036] (4) Ext...

experiment example 3

[0056] Experimental Example 3: Antibacterial test of purified PnPR10-2 recombinant protein

[0057] 1. Preparation of bacteria-carrying plates

[0058] Screen the improved PDA medium for suitable strains of Fusarium solani and Cylindrocarpon destructans, and obtain the best culture conditions. Prepare an improved PDA solid plate (diameter 90 mm), pick mycelium with an inoculation needle, inoculate it on the PDA medium, and culture it in an incubator at 28° C. for 72 hours to prepare a plate with bacteria.

[0059] 2. Inhibitory effect experiment of recombinant PnPR10-2 protein on pathogens

[0060] Antibacterial test was carried out by the paper disc method, 10 μL of purified recombinant PnPR10-2 protein was dropped onto a sterilized filter paper disc with a diameter of 6 mm, and placed on the prepared PDA solid medium plate with plaque for co-cultivation, phosphate buffer saline As a negative control, observe and measure the width of the antibacterial zone of the filter pap...

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Abstract

The invention discloses a radix notoginseng disease resistance-related protein PnPR10-2, and a coding gene and an application thereof. An amino acid sequence of the protein is shown in SEQ ID NO:2, and a nucleotide sequence of the gene for coding the protein is shown in SEQ ID NO:1. The radix notoginseng disease resistance-related protein PnPR10-2 has a wide application prospect in the field of plant resistance to root rot, can be used for culturing root rot-resistant horticultural plants, and has a certain economic benefit potential.

Description

technical field [0001] The invention relates to a Panax notoginseng disease resistance-related protein PnPR10-2, its coding gene and application. Background technique [0002] After Panax notoginseng is infected by pathogenic bacteria, a series of genes in the plant are up-regulated in response, some of which are related to defense responses, including disease process-related proteins (PR) and antibacterial genes, which can enhance their protein activity to Improve disease resistance. PR proteins are considered to be induced by pathogenic bacteria and abiotic stress. According to the structure and function of PR proteins, 17 PR protein families have been identified in monocots and dicots. PR-10 is a PR protein widely distributed in seed plants. It is very similar to tree pollen allergens and food allergens. It belongs to the Bet v 1-like superfamily. The molecular weight of the protein encoded by this gene is 15-19kD, etc. The electric point is slightly acidic, no signal ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/82
CPCC07K14/415C12N15/8205C12N15/8282
Inventor 李昆志杨丹包燚刘文霞张恒丽陈丽梅徐慧妮
Owner KUNMING UNIV OF SCI & TECH
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