Construction and transformation of CRISPR/Cas9 genome editing vector of Flammulina filiformis G protein-coupled receptor gene

LIN Jin-De1,2 YANG Xue-Qin1,2 WEI Tao1,2 GUO Li-Qiong1,2 LIN Jun-Fang1,2 CHEN Yun-Sheng1 HUANG Shi-Shi1,2

(1.College of Food Science, South China Agricultural University, Guangzhou, Guangdong, China 510642)
(2.Research Center for Microecological Agents of Guangdong Province, Guangzhou, Guangdong, China 510642)
【Knowledge Link】G protein-coupled receptor

【Abstract】Two putative G protein-coupled receptor genes Fvgpcr1 and Fvgpcr2 in Flammulina filiformis were obtained by amino acid homology alignment (Blast P) and transcriptome data analysis of the fungus at hypha and primordium stages before and after cold induction. Four expression vectors including pCAMBIA0390-hph-Fvcas9-Fvgpcr1-sgRNA1/sgRNA2 and pCAMBIA0390-hph-Fvcas9-Fvgpcr2-sgRNA1/sgRNA2 were constructed for gene disruption of G-protein coupled receptor genes Fvgpcr1 and Fvgpcr2. The expression vector pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA was introduced into the mycelium of F. filiformis by Agrobacterium tumefaciens-mediated transformation (ATMT). Transformants harvested from hygromycin and cefotaxime rescreening were identified by PCR, RT-qPCR, and Western hybridization. Results showed that the expression vectors pCAMBIA0390-hphFvcas9-Fvgpcr-sgRNA1 and pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA2 were successfully integrated into the genome of F. filiformis and the FvCas9 protein was successfully expressed. However, the Fvgpcr deletion mutant was not obtained. This study used the ATMT method to construct a CRISPR/Cas9 gene disruption system in F. filiformis, which is of great significance for the subsequent gene disruption.

【Keywords】 Flammulina filiformis; CRISPR/Cas9; G protein-coupled receptor; Agrobacterium-mediated transformation;

【DOI】

【Funds】 National Natural Science Foundation of China (31372116, 31572178) Expert of Guangdong Modern Agricultural Edible Fungus Industry System (2018LM1125, 2018LM1126)

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(Translated by HE Z)

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This Article

ISSN:1672-6472

CN: 11-5180/Q

Vol 38, No. 03, Pages 349-361

March 2019

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Abstract

  • 1 Materials and methods
  • 2 Results and analyses
  • 3 Discussion
  • References