cry1A基因通用检测方法的建立  

Development of a Universal Detection Method for cry1A Gene

作　　者：董美[1] 胡晓颖 孟丽霞 宛煜嵩[1] 刘卫晓 金芜军[1] 李亮[1] DONG Mei;HU Xiaoying;MENG Lixia;WAN Yusong;LIU Weixiao;JIN Wujun;LI Liang(Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]中国农业科学院生物技术研究所,北京100081

出　　处：《中国农业科技导报》2020年第7期174-180,共7页Journal of Agricultural Science and Technology

基　　金：国家转基因生物新品种培育重大专项(2016ZX08012-003)。

摘　　要：cry1A基因编码苏云金芽孢杆菌杀虫晶体蛋白,是转基因植物使用最为广泛的抗虫基因,包括cry1Ab、cry1Ac、cry1A.105、cry1Ab/Ac等类型,cry1A基因为靶标的检测方法在转基因植物安全监管与检测中尤为重要。虽已建立数种cry1A基因检测方法,但均因该基因间序列差别较大而无法覆盖大部分转化体。由此,采用简并引物的策略,开发了一种cry1A基因通用检测方法,检测能力覆盖了常见的16种含cry1A的转化体,开发过程包括实验室内部方法建立和实验室间循环验证。结果表明,该方法在引物终浓度0.4μmol·L^-1,退火温度64℃的条件下,能够特异性检测样品中含有的cry1A基因,检出限为0.1%。开发的cry1A基因检测方法参数全面,重复性和重现性良好,为国内外转基因成分监管与检测提供方法参考。cry1A gene encodes Bacillus thuringiensis insecticidal crystal protein,which is the most widely used insect-resistant gene in genetically modified(GM)plants,including cry1Ab,cry1Ac,cry1A.105,cry1Ab/Ac,etc..The detection method on targeting cry1A gene has particularly important in GM plants safety supervision and detection.Several cry1A gene detection method has been developed,but because of much difference among this gene sequences,the detection method can’t cover major GM events.Thus,this paper adopted the strategy of degenerate primers to develop a universal detection method for cry1A gene,detection capability covered 16 GM events.The development process included the establishment of detection method in the internal laboratory and the collaborative validation between laboratories.The results showed that this method could specifically detect the cry1A gene in the samples and limit of detection(LOD)was 0.1%under the condition of 0.4μmol·L^-1 primer concentration and 64℃annealing temperature.The detection method of cry1A gene had comprehensive parameters,good repeatability and reproducibility,which providing a method reference of GMO supervision and detection.

关 键 词：cry1A基因 简并引物 方法建立 成分检测 转基因监管 

分 类 号：Q78[生物学—分子生物学]