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Application of oxathiapiprolin resistant gene RORP1 as selection marker during oomycetes conversion

A fluthiazolidin resistance and fluthiazolidin technology, which is applied in the field of genetic engineering, can solve the problems of limited screening markers, difficulty in re-knockout, and low screening efficiency

Active Publication Date: 2018-04-20
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the selection markers available in oomycetes are very limited at present, and the only reliable selection marker is the NPTII gene, which encodes a protein that can confer resistance to geneticin (G418) in oomycetes, so that transformants can be screened out.
However, when NPT II is used in the CRISPR / Cas9 gene knockout system, the transformant carrying the marker has G418 resistance and cannot be used again for complementation experiments; Knockout of other genes in transformants is still difficult to achieve; in addition, when the NPTII gene is used as a selection marker for the genetic transformation of some oomycetes such as Phytophthora capsici, there are problems such as low selection efficiency

Method used

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  • Application of oxathiapiprolin resistant gene RORP1 as selection marker during oomycetes conversion
  • Application of oxathiapiprolin resistant gene RORP1 as selection marker during oomycetes conversion
  • Application of oxathiapiprolin resistant gene RORP1 as selection marker during oomycetes conversion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1. Application of Screening Marker RORP1 in Gene Complementation of Phytophthora capsici

[0068] Since the sgRNA expressed by the sgRNA expression vector (pYF2.3G-N) used in the transformation of the above plasmid can target the NPTII gene, the vector can be used for complementation of the PcDHCR7 gene knockout transformant of Phytophthora capsici.

[0069] Construct the homology arm vector required for PcDHCR7 gene complementation; simultaneously transform the Cas9 protein expression vector pYF2-PsNLS-hSpCas9 (published vector, Fang and Tyler, 2016, Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR / Cas9 ), using the restriction endonuclease EcoR I to digest the pYF2-PsNLS-hSpCas9 vector, remove the NPT II gene and its promoter and terminator, and then connect with T4 ligase to obtain a Cas9 protein expression vector without the NPT II gene, Named pYF2-Cas9EI. The anteroporative homology arm vector and the...

Embodiment 2

[0077] Example 2, Application of Screening Marker RORP1 in Genetic Transformation of Phytophthora soybean

[0078] In order to expand the scope of application of the screening marker, the above-mentioned plasmid (pYF2.3G-RORP1-N) with the fluthiazolidone resistance gene was transferred into Phytophthora sojae protoplasts to verify whether the screening marker of the present invention can The method is used for the screening of transformants when it is applied to the genetic transformation of Phytophthora sojae, and the work efficiency when the screening marker is used for the genetic transformation of Phytophthora soybean.

[0079] The specific operation process is as follows:

[0080] The wild-type strain of Phytophthora sojae was used as the experimental material, and protoplasts were obtained by referring to the method of Fang et al., and the vector (pYF2.3G-RORP1-N) carrying the fluthiazolidin resistance gene was transferred into the protoplasts.

[0081] The protoplasts ...

Embodiment 3

[0085] Embodiment 3, the application of screening marker RORP1 in the genetic transformation of litchi peronosophthora

[0086] In order to further confirm the scope of application of the screening marker, the above-mentioned plasmid (pYF2.3G-RORP1-N) with the fluthiazolidin resistance gene was transferred into the protoplast of Lychee peronospora, to verify the screening marker of the present invention Whether it can be applied to the screening of transformants during genetic transformation of Pythophthora litchie, and the working efficiency of the screening marker when used for genetic transformation of Pythophthora litchie.

[0087] The specific operation process is as follows:

[0088] The wild-type strain of Phytophthora lychee was used as the experimental material, and protoplasts were obtained by referring to the method of Fang et al., and the vector (pYF2.3G-RORP1-N) carrying the fluthiazolidin resistance gene was transferred into the protoplasts.

[0089] The protopl...

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Abstract

The invention relates to the field of gene engineering, and specifically discloses an application of an oxathiapiprolin resistant gene as a selection marker during oomycetes conversion. A coding geneof mutant protein which is capable of causing oomycetes to generate no less than 300 times resistance to oxathiapiprolin is taken as a selection marker and is used for transformant screening during oomycetes conversion. It is proved through experiments that the positive incidence of transformant screening can reach 100%. The oxathiapiprolin resistant gene can be taken as a selection marker and used for a genetic transformation experiment of oomycetes. Oxathiapiprolin is taken as a screening drug and is high in screening efficiency and screening positive incidence, wide in application range andlow in cost. Through development of the selection marker, the problem that the selection marker is single and the screening efficiency is low during oomycetes conversion is solved, and powerful guarantee is provided with respect to gene anaplerosis and gene knock-out during oomycetes conversion.

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] Oomycetes mainly include Saprolegniales and Peronosporales, which can cause various plant or animal diseases. In the 1840s, Phytophthora infestans in oomycetes caused the famine in Ireland, which had a serious impact on Europe and the whole world, and had a huge impact on human civilization. [0003] Although oomycetes are very similar to fungi in morphology, with the continuous development of molecular biology techniques, people have gradually discovered that they are farther related to fungi and animals (Latijnhouwers et al., 2003; Jiang and Tyler, 2012). In addition to the distant genetic relationship, oomycetes differ from fungi in several ways (Judelson, 2005). For example, in terms of genome size, the genome size of oomycetes is generally between 50-250Mb, while the genome size of fungi ...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/80C12N15/65C12N15/90
CPCC07K14/37C12N15/65C12N15/80C12N15/902C12N2810/10
Inventor 刘西莉王为镇薛昭霖蔡萌张灿苗建强彭钦黄中乔
Owner CHINA AGRI UNIV
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