弓形虫MORN2基因敲除株的构建及其表型鉴定

doi:10.16431/j.cnki.1671-7236.2018.09.005

Abstract

Abstract:

This study was aimed to construct membrane occupation and recognition nexus protein 2 (MORN2) gene knockout strain of Toxoplasma gondii type Ⅰ RH strain,and evaluate the functions of MORN2 gene.The sgRNA target of MORN2 gene was screened to design primers of sgRNA using CRISPR/Cas9 technology.MORN2 CRISPR-targeted plasmid (sgMORN2) was constructed using pSAG1∷CAS9-U6∷sgUPRT as a template under the effect of Q5 enzyme.One pair of homologous primers was designed according to the sgMORN2 target sequence to amplify a DHFR fragment with pUPRT∷DHFR-D plasmid as a template.The MORN2-CRISPR plasmid and the homologous DHFR fragment were cotransfected into Toxoplasma gondii tachyzoites to perform pyrimethamine screening,monoclonal selection in 96 well plates and PCR identification.The results showed that the MORN2 knockout strain (△MORN2) was successfully created.The plaque assays showed that the knockout strain had the same growth rate as the wild strain in vitro plaque assay.Mice were injected intraperitoneally with tachyzoites of 100 and 1 000 knockout strains in vivo infection test,and the death time of mice was basically the same as that of control group.These results indicated MORN2 gene knockout had no effect on the growth rate of Toxoplasma gondii RH strain in vitro and the virulence of mouse infection.This study demonstrated that MORN2 gene had no phenotypic function in RH strain,which laid the foundation for further exploration of the functional genes of Toxoplasma gondii.

Key words: Toxoplasma gondii; CRISPR/Cas9; MORN2 gene; gene knockout

CLC Number:

S852.72⁺3

CHEN Kai, WANG Jinlei, BAI Mengjie, YANG Wenbin, HUANG Siyang. Construction and Phenotype Identification of Toxoplasma gondii MORN2 Gene Knockout Strain[J]. , 2018, 45(9): 2386-2393.

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Construction and Phenotype Identification of Toxoplasma gondii MORN2 Gene Knockout Strain

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