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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
郝振凯¹,汤新明²,张媛媛³,谢福杰¹,陈君敏¹,索静霞¹,索勋¹,刘贤勇^1*
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(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)

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23 992 361×2，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3 106和3 036，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HG994969.1:2 704 213~2 705 255；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。

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

DOI：10.11841/j.issn.1007-4333.2023.08.15

投稿时间：2022-11-10

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

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

HAO Zhenkai¹,TANG Xinming²,ZHANG Yuanyuan³,XIE Fujie¹,CHEN Junmin¹,SUO Jingxia¹,SUO Xun¹,LIU Xianyong^1*

(1.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;2.Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;3.College of Biological Science, China Agricultural University, Beijing 100193, China)

Abstract:

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 23 992 361×2 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 3 106 and 3 036, with the mean coverage depth was 140×. The sequence of chimeric reads indicated that there was only one insertion site, located at HG994969. 1: 2 704 213-2 705 255. 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.

Key words: T-DNA insertion sites Eimeria tenella second-generation sequencing transgene