转基因油菜RF1的外源基因插入位点分析与事件特异性检测方法研究  

Junction sequence of the transgenic rapeseed event RF1 and the methods of qualitative PCR detection

作　　者：聂淑晶[1] 武玉花[1] 吴刚[1] 肖玲[1] 卢长明[1] 

机构地区：[1]中国农业科学院油料作物研究所,湖北武汉430062

出　　处：《中国油料作物学报》2008年第3期265-271,共7页Chinese Journal of Oil Crop Sciences

基　　金：国家863项目(2006AA10Z444);国家公益事业专项资助(2005DIA2J026)

摘　　要：采用基因组步移和巢式PCR方法研究了转基因油菜雄性不育恢复系RF1外源基因插入位点序列特征,并根据此特征建立了RF1转化事件特异性检测方法。从RF1基因组DNA中分离到外源基因插入位点的左边界旁侧序列798bp,包括335bp的插入载体序列和463bp的旁侧基因组序列。根据已发表的RF1右边界旁侧序列的信息,发现外源T-DNA在向油菜基因组整合的过程中,在整合位点处有75bp的基因组序列丢失。根据分离的RF1左边界旁侧序列,建立了RF1事件特异性定性检测方法,扩增片断大小为284bp。利用建立起来的RF1事件特异性定性检测方法可以准确的将转基因油菜RF1与其它的转基因油菜品种区分开,检测灵敏度达到5个拷贝(0.013%)。该事件特异性检测方法具有高度的特异性和良好的灵敏性,为转基因油菜品种RF1的身份识别提供了有效的方法。This paper was designated to discover the integration site of the transgene in the rapeseed genome and to establish event specific methods for qualitative detection of Rfl based on the left border junction fragment, which was isolated using the amended GenomeWalkerTM and the Nested - PCR methods. Sequence alignment between the T - DNA sequence and isolated junction fragments showed that a 798 bp junction fragment of RF1 con- rained 335bp of T -DNA sequence and 463 bp of rapeseed genome DNA, and we found that 75 bp of isolated rapeseed genome DNA sequence were deleted during the integration of the construct. RF1 event - specific qualitative PCR method was established with the primers （RF1LG -F/RF1LV -R） targeting the junction regions to produce a 284 bp product. The qualitative PCR assay showed limits of detection were 5 copies（0.013% ）. The method developed in this study proved to be highly specific, sensitive, and suitable for RF1 sample detection.

关 键 词：GMO 事件特异性检测 定性PCR RF1转化事件 左边界旁侧序列 

分 类 号：S565.4[农业科学—作物学] Q788[生物学—分子生物学]