中国兽医科学2024，Vol.54Issue(6)：742-748,7.DOI:10.16656/j.issn.1673-4696.2024.0107
CRISPR/Cas9技术在构建荧光标记柔嫩艾美耳球虫β-微管蛋白敲入虫株上的应用
Application of CRISPR/Cas9 technology in constructing fluorescent labeled β-tubulin knock in strains of Eimeria tenella
摘要
Abstract
In order to evaluate the application of CRISPR/Cas9 plasmid of Toxoplasma gondii in gene tapping of Eimeria tenella,Etβ-tubulin was used as the target gene.gRNA was designed according to the sequence of Etβ-tubulin gene,and gene-edited plasmids containing gRNA and homologous recombinant plasmids containing target genes and mCherry genes were constructed.The gRNA-containing gene editing plasmid and the homologous recombinant plasmid containing the target gene,the DHFR gene and the mCher-ry gene were co-transfected with E.tenella sporozoites,and the transfected sporozoites were inoculat-ed into the cloaca of chicks,and screened by adding pyrimethamine to the feed,to obtain labeled coc-cidia strains expressing exogenous fluorescent proteins.Single oocysts were selected for propaga-tion,and the labeled strains were identified at gene level and protein level by PCR and fluorescence detection.The results showed that a fluorescent-labeled β-tubulin strain of E.tenella was successfully obtained through single oocyst screening and identification.The results showed that CRISPR/Cas9 tech-nology could be applied to gene knocking in E.tenella,and it also laid a foundation for further study of the function of Et β-tubulin in the parasite.关键词
柔嫩艾美耳球虫/CRISPR/Cas9/基因敲入/β-tubulinKey words
Eimeria tenella/CRISPR/Cas9/knock-in/β-tubulin分类
农业科技引用本文复制引用
肖琴,曲自刚,李文卉,张念章,孙晓林,付宝权..CRISPR/Cas9技术在构建荧光标记柔嫩艾美耳球虫β-微管蛋白敲入虫株上的应用[J].中国兽医科学,2024,54(6):742-748,7.基金项目
宁夏农业农村领域重点研发计划项目(2022BBF02019) （2022BBF02019）
国家自然科学基金项目(32160843) （32160843）
