一种适合转基因棉CpTI和cry1A基因剂量测定的标准质粒的构建和应用  

Construction and Application of a Reference Plasmid Suitable for Determination of CpTI and cry1A Gene Dosages in Genetically Modified Cottons

作　　者：苏长青[1,2] 谢家建[1] 孙爻[1] 彭于发[1] 

机构地区：[1]植物病虫害生物学国家重点实验室,农业部转基因植物环境安全监督检验测试中心(北京),中国农业科学院植物保护研究所,北京100193 [2]衡水学院生命科学系,河北衡水053000

出　　处：《作物学报》2011年第9期1533-1539,共7页Acta Agronomica Sinica

基　　金：国家重点基础研究发展计划(973计划)项目(2007CB109201);国家转基因新品种培育重大科技专项(2011ZX08012-002,2011ZX08011-001)资助

摘　　要：在转基因检测领域,标准质粒以容易获得、纯度高、成本低和稳定性好的优点逐渐被广泛应用,适合同时检测多个靶标基因的需求。本研究针对我国抗虫棉(Gossypium hirsutum L.)的主要外源基因类型,构建含有豇豆胰蛋白酶抑制剂基因(CpTI)、苏云金杆菌晶体杀虫蛋白基因(cry1A)和棉花内标准基因硬脂酰-酰基载体蛋白脱饱和酶基因(Sad1)的多靶标质粒pMD-CCS作为标准物质,建立了CpTI和cry1A基因的实时荧光定量PCR方法。测定了我国9个抗虫棉品种中的CpTI和cry1A基因剂量,显示科棉3号等3个抗虫棉品种中CpTI基因的平均剂量为0.020～0.018拷贝/基因组,cry1A基因的平均剂量为1.377～2.136拷贝/基因组;鄂杂棉1号F1等6个抗虫棉品种中cry1A基因的平均剂量为0.887～2.564拷贝/基因组,定量结果的标准差(SD)范围在0.001～0.149之间。结果说明,pMD-CCS适合作为标准物质用于抗虫棉中CpTI和cry1A基因的定量测定。In transgenic detection field,a preferable reference material（RM） has been developing with the advantage of easy availability,high purity,low cost and good stability,which is more suitable for detecting multiple target genes.In the present study,we constructed a multi-target reference plasmid named pMD-CCS containing cowpea trypsin inhibitor（CpTI） and Bacillus thuringiensis insecticidal crystal protein（cry1A） and cotton endogenous gene Stearoyl-acyl carrier protein desaturase（Sad1） sequences targeting the key exogenous gene types of the insect resistant cotton varieties（Gossypium hirsutum L.） in China.The real-time quantitative PCR methods for CpTI and cry1A were established using pMD-CCS as the RM.The dosages of CpTI and cry1A from nine insect resistant cotton varieties were determined.The average CpTI dosages were 0.020-0.018 copies/genome and the average cry1A dosages were 1.377-2.136 copies/genome in three insect resistant cotton varieties including Kemian3.The average cry1A dosages were 0.887-2.564 copies/genome in six ones including Ezamian1-F1.The standard deviations（SD） of the quantitative measurement ranged from 0.001-0.149.The above results demonstrated that pMD-CCS could be used as the RM for the quantitative measurement of CpTI and cry1A genes in insect resistant cotton varieties.

关 键 词：抗虫棉 CPTI基因 crylA基因 基因特异性实时PCR 标准质粒 

分 类 号：S562[农业科学—作物学]