利用二代测序技术对柔嫩艾美耳球虫T-DNA整合位点的分析及鉴定  

Analysis and identification of T-DNA insertion sites in transgenic Eimeria tenella

作　　者：郝振凯 汤新明 张媛媛 谢福杰 陈君敏 索静霞 索勋[1] 刘贤勇 HAO Zhenkai;TANG Xinming;ZHANG Yuanyuan;XIE Fujie;CHEN Junmin;SUO Jingxia;SUO Xun;LIU Xianyong(College of Veterinary Medicine/National Key Laboratory of Veterinary Public Health Security/Key Laboratory ofAnimal Epidemiology and Zoonosis of Ministry of Agriculture,China Agricultural University,Beijing 100193,China;Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100193,China;College of Biological Science,China Agricultural University,Beijing 100193,China)

机构地区：[1]中国农业大学动物医学院/兽医公共卫生安全全国重点实验室/农业农村部动物流行病学重点实验室,北京100193 [2]中国农业科学院北京畜牧兽医研究所,北京100193 [3]中国农业大学生物学院,北京100193

出　　处：《中国农业大学学报》2023年第8期183-190,共8页Journal of China Agricultural University

基　　金：国家重点研发计划(2018YFD0500300,2016YFD0501300);国家自然科学基金项目(32072884,31873007)。

摘　　要：为建立一种适用于转基因球虫的快速且准确的分析T-DNA整合位点的方法,本研究利用二代测序技术对转基因柔嫩艾美耳球虫EtM2e虫株进行基因组重测序分析,基于嵌合体reads比对分析T-DNA的插入位点,通过T-DNA特有序列以及外源基因与球虫同源序列的测序深度进而分析T-DNA拷贝数,最后利用PCR扩增验证分析位点。结果表明:(1)二代测序下机数据过滤后获得数据7.2 Gb,Q20为96.1%,Q30为89.2%,能够比对至柔嫩艾美耳球虫参考基因组的reads数为23992361×2,平均测序深度为80×,reads全基因覆盖结果表明整个染色体被覆盖的较为均匀,测序的随机性比较好,可以进行后续分析;(2)比对至T-DNA序列的reads数为3106和3036,平均测序深度为140×,嵌合体reads序列信息表明T-DNA只存在一个插入位点,位于HG994969.1:2704213~2705255;(3)T载体骨架特有序列卡那抗性基因的平均测序深度为67×,T-DNA特有序列EYFP基因的平均测序深度为85×,而T-DNA与球虫同源序列His4基因的5′调控区序列的平均测序深度为154×,Actin基因的3′调控区序列的平均测序深度为164×,同源序列的平均测序约为特有序列平均测序深度的2倍,这表明T-DNA为单拷贝,与发现一个插入位点的结果相符合;(4)经PCR验证分析成功鉴定了该整合位点。转基因艾美耳球虫EtM2e虫株T-DNA整合位点的鉴定为后续转基因球虫鉴定提供了技术平台,也为球虫活载体疫苗的研发提供了一个潜在的靶位点。To establish a rapid and accurate method for the analysis of T-DNA integration sites in transgenic coccidia,in this study,EtM2e,a transgenic E.tenella strain,was sequenced with whole-genome resequencing.The insertion site of T-DNA was analyzed by chimeric reads and the T-DNA copy number was analyzed by sequencing depth of T-DNA unique sequences and homologous sequences with Eimeria tenella.The insertion site was further validated by PCR amplification.The results showed that(1)7.2 Gb of clean data were obtained after filtering.Q20 was 96.1%and Q30 was 89.2%,with the number of matched reads was 23992361×2 and mean coverage depth was 80×.The whole genome coverage of reads showed the chromosomes were covered evenly and the randomness of sequencing was good.(2)The number of matched reads mapped to the T-DNA sequence was 3106 and 3036,with the mean coverage depth was 140×.The sequence of chimeric reads indicated that there was only one insertion site,located at HG994969.1:2704213-2705255.(3)The mean coverage depth of T-vector backbone unique sequence Kanamycin and EYFP were 67×and 85×,while the mean coverage depth of homologous sequence of the 5′regulatory region of E.tenella His 4 and 3′regulatory region of Actin was 154×and 167×.The average sequencing of the homologous sequence was about twice of the unique sequence,which indicated that the T-DNA was single copy and was consistent with the result of finding one insertion site.(4)The integration site was successfully identified by PCR validation analysis.The success of validation of T-DNA insertion site in transgenic E.tenella EtM2e strain provides a technique for the identification of other transgenic Eimeria parasites,as well as providing a potential genomic safe harbor for the subsequent anticoccidial vaccine vector development.

关 键 词：T-DNA整合位点 柔嫩艾美耳球虫 二代测序技术 转基因 

分 类 号：S8559.1[农业科学—临床兽医学]