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

作　　者：肖琴 曲自刚[2] 李文卉 张念章[2] 孙晓林[1] 付宝权[2,3] XIAO Qin;QU Zigang;LI Wenhui;ZHANG Nianzhang;SUN Xiaolin;FU Baoquan(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory for Animal Disease Control and Prevention/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院、兰州兽医研究所、动物疫病防控全国重点实验室,甘肃兰州730046 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2024年第6期742-748,共7页Chinese Veterinary Science

基　　金：宁夏农业农村领域重点研发计划项目(2022BBF02019);国家自然科学基金项目(32160843)。

摘　　要：为评价弓形虫CRISPR/Cas9质粒在柔嫩艾美耳球虫基因敲入中的应用,以柔嫩艾美耳球虫Etβ-tubulin为目标基因,根据Etβ-tubulin基因序列设计向导RNA(g RNA),构建含g RNA的基因编辑质粒及含有目标基因和m Cherry基因的同源重组质粒;将含g RNA的基因编辑质粒和含有目标基因、DHFR基因和m Cherry基因的同源重组质粒共同转染柔嫩艾美耳球虫子孢子,将转染后的子孢子接种雏鸡泄殖腔,并在饲料中添加乙胺嘧啶进行筛选,得到表达外源荧光蛋白的标记球虫虫株。挑取单卵囊进行扩繁,并通过PCR和荧光检测方法分别从基因水平和蛋白水平上对标记虫株进行了鉴定。结果显示通过单卵囊筛选和鉴定,成功获得了1株荧光标记β-微管蛋白的柔嫩艾美耳球虫虫株。结果表明,CRISPR/Cas9技术可应用到柔嫩艾美耳球虫的基因敲入研究,同时也为进一步研究Etβ-tubulin在虫体中的功能奠定了基础。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.g RNA was designed according to the sequence of Etβ-tubulin gene,and gene-edited plasmids containing g RNA and homologous recombinant plasmids containing target genes and m Cherry genes were constructed.The g RNA-containing gene editing plasmid and the homologous recombinant plasmid containing the target gene,the DHFR gene and the m Cherry gene were co-transfected with E.tenella sporozoites,and the transfected sporozoites were inoculated into the cloaca of chicks,and screened by adding pyrimethamine to the feed,to obtain labeled coccidia strains expressing exogenous fluorescent proteins.Single oocysts were selected for propagation,and the labeled strains were identified at gene level and protein level by PCR and fluorescence detection.The results showed that a fluorescent-labeledβ-tubulin strain of E.tenella was successfully obtained through single oocyst screening and identification.The results showed that CRISPR/Cas9 technology could be applied to gene knocking in E.tenella,and it also laid a foundation for further study of the function of Etβ-tubulin in the parasite.

关 键 词：柔嫩艾美耳球虫 CRISPR/Cas9 基因敲入 Β-TUBULIN 

分 类 号：S852.723[农业科学—基础兽医学]