转基因水稻E1C9K-18的获得及其事件特异性检测方法的建立  

Development of Transgenic Rice(Oryza sativa)E1C9K-18 and Establishment of Its Event-specific Detection Method

作　　者：徐礼逵 邓力华[1] 李华 肖国樱[1,3] XU Li-Kui;DENG Li-Hua;LI Hua;XIAO Guo-Ying(Key Laboratory of Agro-ecological Processes in Subtropical Region,Institute of Subtropical Agriculture,Chinese Academy of Sciences,Changsha 410125,China;University of Chinese Academy of Sciences,Beijing 100049,China;Innovation Academy for Seed Design,Chinese Academy of Sciences,Beijing 100101,China)

机构地区：[1]中国科学院亚热带农业生态研究所/亚热带农业生态过程重点实验室,长沙410125 [2]中国科学院大学,北京100049 [3]中国科学院种子创新研究院,北京100101

出　　处：《农业生物技术学报》2020年第12期2250-2260,共11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(2016ZX08001003-001)。

摘　　要：抗除草剂、抗螟虫水稻在方便杂草治理、减少虫害损失方面具有重要的应用价值。事件特异性检测方法是监管转基因水稻的必需技术。本研究构建了损伤诱导启动子驱动抗螟虫基因杀虫蛋白基因(gene coding insecticidal crystal protein Cry1Ca,Cry1Ca#)和组成型启动子驱动抗除草剂基因5-烯醇丙酮酰莽草酸-3-磷酸合成酶基因(gene coding 5-enolpyruvylshikimate 3-phosphate synthase,Epsps#)的植物表达载体,利用农杆菌(Agrobacterium tumefaciens)介导法转化籼稻(Oryza sativa subsp.indica)9K19-5,获得了单拷贝转化事件E1C9K-18,喷施草甘膦显示其具有良好的草甘膦抗性。利用高效热不对称交错PCR法(high efficient thermal asymmetric interlaced PCR,hiTAIL-PCR)扩增获得了转基因水稻E1C9K-18外源基因插入位点的右旁侧序列420 bp,其插在水稻基因组第4号染色体长臂的33189510位核苷酸残基之后。根据水稻参考基因组序列和T-DNA的左侧序列,设计引物扩增得到了613 bp的左旁侧序列,其插在水稻基因组第4号染色体长臂的33189480位核苷酸残基之前。外源基因插入导致水稻基因组上缺失了29个核苷酸残基。基于左、右旁侧序列,建立了转基因水稻E1C9K-18的事件特异性定性PCR检测方法,以及快速鉴定转基因后代植株外源基因型的三引物PCR检测方法。事件特异性PCR和三引物PCR检测方法将为转基因水稻E1C9K-18的研究和应用提供技术支撑。The herbicide resistance and Lepidopteran resistance of rice(Oryza sativa)play important roles in weeds management and reduction of yield loss by insect pests,and the event-specific detection method is an essential technique for supervision of transgenic rice.In this study,the expression vector was constructed firstly,in which the Lepidopteran resistance gene Cry1 Ca#(gene coding insecticidal crystal protein Cry1 Ca)was driven by wound-induced promoter and the herbicide resistance gene Epsps#(gene coding 5-enolpyruvylshikimate 3-phosphate synthase)was driven by constitutive promoter,and a single copy transgenic event E1 C9 K-18 was developed by Agrobacterium-mediated genetic transformation from 9 K19-5(O.sativa subsp.indica),which possessed elite glyphosate resistance.Then,the hiTAIL-PCR(high-efficiency thermal asymmetric interlaced PCR)was adopted to reveal the flanking sequence of transgenic rice E1 C9 K-18,and the right flanking sequence with 420 bp length was discovered.Comparing the right flanking sequence with rice genome database,it was found the exogenous fragment was inserted behind the nucleotide residue No.33189510 of chromosome 4.The left flanking sequence with 613 bp length was amplified using the primers that were designed according to the sequence of integration site on rice genome and left side sequence of T-DNA.Compared the left flanking sequence with rice genome database,it was found the exogenous fragment was inserted before the nucleotide residue No.33189480 of chromosome 4.However,the insertion of T-DNA resulted in a deletion of 29 nucleotide residues in rice genome.Based on the left and right flanking sequences,the event-specific PCR detection method and tri-primers PCR method were developed for E1 C9 K-18,which will provide technical supports for identification of this transgenic event and rapidly selection of homozygote from segregation population.

关 键 词：转基因水稻 旁侧序列 事件特异性检测 基因型鉴定 

分 类 号：S-3[农业科学]