摘要
大豆疫霉根腐病是由卵菌纲病原菌—疫霉菌(Phytophthora sojae)引起的大豆毁灭性病害之一。本研究前期利用农杆菌介导转化技术,将广谱抗病基因hrpZm导入栽培大豆Williams 82,获得高抗疫霉根腐病转基因大豆新品系B4J8049,并已进入环境释放试验阶段。为进一步推进该转化事件的生物安全评价及应用,本研究采用热不对称交错PCR(thermal asymmetric interlaced PCR,TAIL-PCR)方法分析外源T-DNA整合位点右边界序列,并依据其序列特征,建立该转化事件特异性检测方法。Southern杂交检测结果表明,转基因大豆事件B4J8049外源T-DNA插入拷贝数为1个。根据外源T-DNA插入片段序列设计3条嵌套特异性检测引物,与简并引物组合进行TAIL-PCR扩增反应,获得外源TDNA插入位点右边界序列。BLAST分析(https://soybase.org/)表明,外源T-DNA片段以单拷贝形式反向插入的方式整合到大豆基因组中第8号染色体的187 824位点。在此基础上,依据插入位点右边界序列设计检测引物,建立转基因大豆事件B4J8049特异性检测方法。本研究为该抗病转基因事件B4J8049及其衍生产品特异性检测提供依据。
Phyt0phthora root and stem rot, caused by an oomycete pathogen Phytophthora sojae, is one of the mostdevastating diseases in soybean production. In our previous study, we introduced a broad-spectrum disease resistantgene hrpZm into the soybean cultivar ＇Williams 82＇by the Agrobacterium-mediated transformation to generate thetransgenic event B4 J8049,which exhibited high resistance to P. sojae and approved for environmental release. Toadvance safety assessment and application of the transgenic soybean event, we analyzed the insertion site of the for-eign T-DNA in the genome of the event B4 J8049 using the thermal asymmetric interlaced PCR（TAIL-PCR）, andestablished the event-specific detection method based on the flanking sequence at the right border of the T-DNA in-sertion. Southern blot analysis confirmed the single-copy insertion of the foreign T-DNA in the soybean genome.Three nested specific primers based on the T-DNA sequence and the arbitrary degenerate primers were designed toamplify the right border flanking sequence by TAIL-PCR. BLAST analysis（https：//soybase.org/） revealed that theforeign T-DNA was inserted into the position 187 824 on the soybean chromosome 8 in the reverse way. Based onthe flanking sequence of the right border, an event-specific detection method was established for the transgenic soy-bean B4 J8049 and its derivative products.
作者
包婉莹
仲晓芳
杜茜
赵倩倩
杨向东
BAO Wanying;ZHONG Xiaofang;DU Qian;ZHAO Qianqian;YANG Xiangdong(School of Life Science,Jilin Normal University,Siping 136000;Agricultural Biotechnology Institute,Jilin Academy of Agricultural Sciences/Jilin Provincial Key Laboratory of Agricultural Biotechnology,Changchun 130033,China)
出处
《东北农业科学》
北大核心
2018年第5期21-26,共6页
Journal of Northeast Agricultural Sciences
基金
国家转基因生物新品种培育科技重大专项(2016ZX08004-004)
吉林省农业科学院创新工程项目(C6215000220
C7208000307)
