基因组测序技术解析耐除草剂转基因水稻G2-7的分子特征  

Molecular characterization identification by genome sequencing of transgenic glyphosate-tolerant rice G2-7

作　　者：马硕 焦悦 杨江涛[1] 王旭静[1] 王志兴[1] MA Shuo;JIAO Yue;YANG Jiang-Tao;WANG Xu-Jing;WANG Zhi-Xing(Biotechnology Research Institute,Chinese Academy of Agricultural Sciences/MARA Key Laboratory on Safety Assessment Molecular of Agri-GMO,Beijing 100081,China;Development Center for Science and Technology/MARA,Beijing 100122,China)

机构地区：[1]中国农业科学院生物技术研究所/农业农村部农业转基因生物安全评价(分子)重点实验室,北京100081 [2]农业农村部科技发展中心,北京100122

出　　处：《作物学报》2020年第11期1703-1710,共8页Acta Agronomica Sinica

基　　金：国家转基因新品种培育重大专项(2016ZX08010-003)资助。

摘　　要：外源DNA片段的拷贝数及插入位点的侧翼序列等分子特征信息是转基因植物安全评价过程中必需要提供的信息。本研究利用基因组测序结合生物信息学对耐除草剂转基因水稻G2-7的T-DNA插入位点、拷贝数和侧翼序列进行鉴定。利用Illumina NovaSeq 6000平台对G2-7进行全基因组测序,共获得47.13 Gb的测序数据,通过与转基因载体和参考基因组序列的比较,确定了G2-7中T-DNA在受体基因组中的插入位点。结果显示,外源DNA片段以单位点单拷贝形式插入到水稻1号染色体的36,189,491~36,189,507位置,造成水稻基因组16 bp DNA缺失,无载体骨架的插入。同时我们获得外源基因插入位点5′侧翼序列375 bp和3′端侧翼序列353 bp,并通过PCR扩增和Sanger测序进一步证明获得的侧翼序列是正确的。研究结果为转基因水稻G2-7的安全评价及转化体特异性检测提供了有效的数据支撑,同时也证明全基因组测序(WGS)是解析转基因植物分子特征的有效方法。Molecular characterization,such as copy number and flanking sequence of foreign DNA fragment insertion site,is the important identity information,provided during safety assessment of genetic modified crop.In this study,the T-DNA insertion site,copy number and flanking sequences were identified in transgenic glyphosate-tolerant rice G2-7 based on whole genome sequencing in combination bioinformatics analysis method.47.13 Gb clean sequence data for G2-7 was generated on Illumina NovaSeq 6000 platform.The junction reads mapped to boundaries of T-DNA and flanking sequences in G2-7 were identified by comparing with sequence of transformation vector and rice reference genome.The results showed that exogenous T-DNA fragments was integrated in the position of Chr.136,189,491–36,189,507 with a single copy,16 bp rice genome sequence was deleted at the insertion site and no insertion of vector backbone.375 bp and 353 bp flanking host DNA sequence of 5′-end and 3′-end of the insertion DNA fragment were also obtained,respectively.The putative insertion location and flanking sequences were further confirmed by PCR amplification and Sanger sequencing.The results not only provided data support for safety assessment and event specific detection,but also demonstrated that WGS was an effective technique for identifying molecular characterization in rice.

关 键 词：基因组测序 转基因水稻 分子特征 拷贝数 侧翼序列 

分 类 号：S511[农业科学—作物学]