基于基因组重测序的高含量油酸转基因大豆T-DNA旁侧序列分析及事件特异性PCR检测  

Analysis of the T-DNA Flanking Sequences and Event-specific PCR Detection of High-content Oleic Acid Transgenic Soybean(Glycine max) Based on Genome Re-sequencing

作　　者：仲晓芳[1] 杨静[1] 贺红利[1] 牛陆[1] 邢国杰[1] 杨向东[1] ZHONG Xiao-Fang;YANG Jing;HE Hong-Li;NIU Lu;XING Guo-Jie;YANG Xiang-Dong(Jilin Provincial Key Laboratory of Agricultural Biotechnology/Agro-Biotechnology Institute,Jilin Academy of Agricultural Sciences,Changehun 130033,China)

机构地区：[1]吉林省农业科学院农业生物技术研究所/吉林省农业生物技术重点实验室,长春130033

出　　处：《农业生物技术学报》2018年第12期2017-2026,共10页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育科技重大专项(No.2016ZX08004-003);吉林省农业科学院创新工程项目(No.c6215000220和No.c7208000307);国家自然科学基金(No.31671764)

摘　　要：基于外源T-DNA与植物基因组的连接区域的特异性建立的特异性检测方法,具有非常高的特异性和准确性,是实现对该转基因事件及其衍生品种的有效监督管理、保障转基因产业健康发展的重要技术手段。本研究前期利用反义RNA和RNAi技术,获得2个油酸含量达78%以上的高油酸转基因大豆(Glycine max)事件。由于T-DNA携带大豆内源基因,旁侧序列不易分析,且为进一步推进上述转化事件的安全评价及应用,本研究基于基因组重测序技术并结合PCR,分析上述2个转基因大豆事件外源TDNA整合位点旁侧序列。Southern杂交结果显示,2个高油酸转基因大豆事件E2D9050和EB8072外源T-DNA插入拷贝数分别为1个和2个。采用BWA-Burrows-Wheeler Alignment tool (http://bio-bwa.sourceforge.net/)方法,将基因组重测序分析获得的转基因事件序列信息与参考大豆基因组进行对比。结果表明,转基因大豆事件E2D9050整合位点为Chr04号染色体的51410941位点,整合方式为反向单拷贝插入;EB8072整合位点为大豆基因组Chr19染色体38147218位点,整合方式为单位点双拷贝反向插入,两个拷贝的右边界相接。结合PCR扩增,分别获得E2D9050和EB8072转基因事件整合位点的左、右边界旁侧序列。依据其序列特征,建立了转基因大豆事件两个转化体特异性检测方法,检测灵敏度为0.1%,为上述2个高油酸转基因大豆事件及其衍生产品特异性检测提供依据。The specific detection method based on the specificity of the connection area between external T-DNA and plant genome has very high specificity and accuracy,which is an important technical means to effectively supervise and manage the transgenic event and its derivatives,and ensure the healthy development of the transgenic industry.In the previous study,2transgenic soybean (Glycine max)events were obtained with >78%oleic acid content.To isolate the flanking sequences of T-DNAs with endogenous genes and promote the safety assessment and application of these 2transgenic events,genome re-sequencing combined with PCR was carried out to analyze the flanking sequences of T-DNAs of these 2transgenic events.There were 1and 2copies T-DNAs in these 2transgenic events by Southern blot analysis,respectively.Using genome re-sequencing compared to reference genome information based on BWA-Burrows-Wheeler Alignment tool method (http://bio-bwa.sourceforge,net/),flanking sequences of T-DNAs in the transgenic soybean ‘E2D9050'and ‘EB8072'genomes were isolated.The T-DNA of ‘E2D9050'was integrated into the position 51410941on Chr04with one copy.The T-DNA of‘EB8072'was integrated into soybean genome with 2 copies in position 38147218on Chr19 and the pattern of integration was inverted insertion with right borders of these copies interfaced.These results were in accord with the results of Southern blot analysis.According to the flanking sequences of these positions,primers were designed and PCR was amplified to verify these flanking sequences of insertion sites.Event-specific primers were designed based on these fragments and the sensitivities of these pairs of primer were both 0.1%.It provides an accurate and fast method for identification of these kinds oftransgenic event.

关 键 词：高油酸转基因大豆 基因组重测序 旁侧序列 事件特异性PCR 灵敏度 

分 类 号：S565.1[农业科学—作物学]