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Application of carboxylic acid amide fungicide resistance gene as screening marker for oomycete transformation

A technology of carboxylic acid amides and resistance genes, which is applied in the field of genetic engineering and can solve problems such as restricting gene complementation and multiple knockouts of Phytophthora capsici

Active Publication Date: 2020-12-01
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the selection markers currently available for genetic transformation of Phytophthora capsici are limited to the NPTⅡ gene with geneticin (G418) resistance, which seriously restricts the development of gene complementation and multiple knockouts of Phytophthora capsici.

Method used

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  • Application of carboxylic acid amide fungicide resistance gene as screening marker for oomycete transformation
  • Application of carboxylic acid amide fungicide resistance gene as screening marker for oomycete transformation
  • Application of carboxylic acid amide fungicide resistance gene as screening marker for oomycete transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Application of screening marker RCesA3 in gene complementation of Phytophthora capsici

[0059] Since the sgRNA expressed by the sgRNA expression vector (pYF2.3G-N) used for the above-mentioned plasmid transformation can target the NPTII gene, the vector can be used for complementation of the above-mentioned Phytophthora capsici PcDHCR7 gene knockout transformant.

[0060] Construct the homology arm vector required for PcDHCR7 gene complementation; transform the Cas9 protein expression vector at the same time: use the restriction endonuclease EcoR I to digest the Cas9 protein expression vector pYF2-PsNLS-hSpCas9 (published vector, Fang andTyler, 2016, Efficient Disruption and replacement of an effector gene in theoomycete Phytophthora sojae using CRISPR / Cas9), the NPTII gene and its promoter and terminator were removed, and then ligated with T4 ligase to obtain a Cas9 protein expression vector without the NPTII gene, which was named pYF2- Cas9EI. The anterop...

Embodiment 2

[0067] Example 2, the extraction of Phytophthora capsici DNA and the verification of gene complementation

[0068] (1) Phytophthora capsici DNA extraction

[0069] Scrape an appropriate amount of mycelium of Phytophthora capsici, put it in a 2mL centrifuge tube, and add 1 steel ball;

[0070] Add 150 μL of extraction buffer (0.35M sorbitol, 0.1M Tris, 0.005M EDTA[pH 7.5], 0.02M sodium bisulfite), shake on the shaker for 30s;

[0071] Add 150 μL of nuclear lysis buffer (0.2M Tris, 0.05M EDTA [pH 7.5], 2.0M NaCl and 2% CTAB [pH 7.5]) and 60 μL of 20% SDS, fully shake (1min) on the shaker and place at 65°C Water bath 30min;

[0072] Add an equal volume (360 μL) of chloroform:isoamyl alcohol (v / v, 24:1) and invert up and down 6-8 times to mix well, and centrifuge at 12000 rpm for 10-20 min;

[0073] Take the supernatant in a new 1.5mL pointed centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (v / v, 24:1) to extract again;

[0074] After centrifugation, take the...

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Abstract

The invention relates to the field of gene engineering, and specifically discloses an application of taking a carboxylic acid amides bactericide resistant gene as an oomycetes conversion screening marker. According to the application, coding gene of a mutant protein, which can make oomycetes generate more than 100 times of resistance on a carboxylic acid amides bactericide, is taken as the screening marker and is used to screen the converter in the process of oomycetes conversion. The screening marker can solve the problem that the screening marker is single in genetic transformation of phytophthora capsici and helps the research on functions of gene of phytophthora capsici.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a novel screening marker for oomycete transformation. Background technique [0002] Phytophthora capsici belongs to Kingdom Chromista, Phylum Oomycota, Class Oomycetes, Order Peronosporales, Family Peronosporaceae, Genus Phytophthora ( Lamour et al., 2012), the pepper blight caused by this pathogen has a short incubation period and rapid development, which is one of the devastating diseases in pepper production. In 1918, the outbreak of chili blight caused serious economic losses to chili production in Chile. Subsequently, the disease was first reported in New Mexico in 1922 (Leonian, 1922), and the disease is now widely occurring around the world. The onset symptoms of pepper blight are closely related to pepper varieties, infected parts and environmental conditions. Usually, the disease occurs more seriously under high temperature and high humidity conditions. The main symp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/65C12N1/15C12Q1/04C12R1/645
CPCC12N15/65C12Q1/04
Inventor 刘西莉王为镇蔡萌薛昭霖张灿李腾蛟方媛刘鹏飞
Owner CHINA AGRI UNIV
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