抗除草剂大豆GE-J12实时荧光定量PCR检测方法的建立  

Establishment of Real-time Fluorescent Quantitative PCR Detection Method for Genetically Modified Herbicide-tolerant Soybean GE-J12

作　　者：曹英芳 赵新 刘双 李瑞环 刘娜 徐石勇 高芳瑞 马卉[3] 兰青阔 檀建新[1] 王永 CAO Ying-fang;ZHAO Xin;LIU Shuang;LI Rui-huan;LIU Na;XU Shi-yong;GAO Fang-rui;MA Hui;LAN Qing-kuo;TAN Jian-xin;WANG Yong(College of Food Science and Technology,Hebei Agricultural University,Baoding 071001;Institute of Germplasm Resources and Biotechnology,Tianjin Academy of Agriculture Science,Tianjin 300381;Rice Research Institute,Anhui Academy of Agricultural Sciences,Hefei 230041)

机构地区：[1]河北农业大学食品科技学院,保定071001 [2]天津农业科学院种质资源与生物技术研究所,天津300381 [3]安徽省农业科学院水稻研究所,合肥230041

出　　处：《生物技术通报》2022年第7期146-152,共7页Biotechnology Bulletin

基　　金：转基因生物新品种培育重大专项(2019ZX08004001);安徽省农业科学院院立科研项目(2021YL006)。

摘　　要：GE-J12是中国农业科学院研发的转G2-EPSPS基因和GAT基因的抗除草剂大豆新品系。根据抗除草剂大豆GE-J12品系外源基因插入位点5’端转化体特异性序列设计引物和探针,经引物探针筛选、特异性测试、反应体系和程序优化、标准曲线构建、检测极限和定量极限测试等试验,建立了基于TaqMan水解探针的实时荧光定量PCR检测方法。该方法特异性强、准确度高,在RSD小于25%的情况下检测极限为0.032%,定量极限为0.16%,线性度大于0.992,经5%、3%和1%定值样品测试,与真实值平均偏差为2.87%-16.67%。可用于转基因耐除草剂大豆GE-J12转化体特异性序列含量的精准定量,为GE-J12大豆新品系的检测提供了新方法。GE-J12 is a new herbicide-resistant soybean line with G2-EPSPS gene and GAT gene developed by the Chinese Academy of Agricultural Sciences.In this study,primers and probes were designed based on the specific sequence of the transformant at the 5’ end of the insertion site of the exogenous gene in the herbicide-resistant soybean GE-J12 strain.A realtime fluorescent quantitative PCR detection method based on TaqMan hydrolysis probe was established,after primer probe screening,specificity testing,reaction system and program optimization,and standard curve construction,detection limit and quantitative limit test and other tests.This method presents strong specificity and high accuracy.When the RSD was < 25%,the detection limit was 0.032%,the quantification limit was 0.16%,and the linearity was > 0.992.After testing with 5%,3%,and 1% fixed-value sample,the average deviation of the true value was 2.87%-16.67%.This method can be used to accurately quantify the specific sequence content of genetically modified herbicide-tolerant soybean GE-J12 transformants,which provides a new method for the detection of new soybean lines of GE-J12.

关 键 词：实时荧光定量PCR GE-J12转化体 特异性序列 

分 类 号：S565.1[农业科学—作物学]