利用多重荧光定量PCR检测转基因水稻外源基因拷贝数  

Determination of the Copy Number of Exogenous Gene in Transgenic Rice(Oryza sativa) by Multiplex qPCR

作　　者：魏毅东[1,2,3] 毛小辉 何炜[1,2,3] 王进兰 连玲[1,2,3] 许秀宝 林悦龙 谢华安[1,2,3] 张建福 

机构地区：[1]福建省农业科学院水稻研究所,福州350019 [2]农业部华南杂交水稻种质创新与分子育种重点实验室/福州(国家)水稻改良分中心/福建省作物分子育种工程实验室/福建省水稻分子育种重点实验室/福建省物种质创新与分子育种省部共建国家重点实验室培育基地/杂交水稻国家重点实验室华南研究基地,福州350003 [3]水稻国家工程实验室,福州350003

出　　处：《农业生物技术学报》2017年第12期2072-2078,共7页Journal of Agricultural Biotechnology

基　　金：国家转基因农作物新品种培育重大专项(No.2016ZX08001-006和No.2016ZX08001-004);福建省财政专项-福建省农业科学院创新团队PI项目(No.2016PI-15)

摘　　要：转基因植物中外源基因的拷贝数影响遗传稳定性和基因表达水平,因此鉴定和筛选单拷贝纯合的植株对于子代材料的功能分析是十分必须的。为了快速、准确的对含有潮霉素抗性基因-潮霉素磷酸转移酶II(hygromycin phosphotransferase II,hpt II)的转基因水稻材料进行拷贝数鉴定,本研究选用了稳定的组成型基因水稻转录剪切因子U2af(Oryza sativa splicing factor U2af,Os SFu2a)作为内参基因,构建基于Taqman的多重荧光定量PCR(Multiplex q RT-PCR)的拷贝数鉴定体系。通过不同引物组合进行扩增效率比较,确定了hpt II-1和Os SFu2a-2为最佳引物组合。利用该体系对10个转基因克隆进行了拷贝数鉴定,结果其中7个克隆为单拷贝植株,3个克隆为双拷贝植株。随后,通过Southern blot和转基因子代分离比统计,验证了此方法的可靠性;并通过与传统的单重荧光定量PCR(Singlex q RT-PCR)法进行比较,结果表明多重荧光定量PCR法大大提高了检测的准确性和稳定性。因此,该方法能够帮助科研人员快速,准确,高通量的鉴定水稻外源基因拷贝数与纯合体植株。The copy number of exogenous gene affects the genetic stability and gene expression level in transgenic plants, and the identification and screening of single copy homozygous plants are necessary for the functional analysis of progeny. To fast and accurately identify gene copy of transgenic rice materials harboring hygromycin resistant gene（hygromycin phosphotransferase II（hpt II）, Taqman based Multiplex qRT-PCR was developed using constructive gene Oryza sativa splicing factor u2af（OsSFu2a） as reference gene. By comparison of amplification efficiency of different primer sets, the primer set of hpt II-1 and OsFU2a-2 was the best one. Thecopy number of 10 transgenic clones was determinated using this system, and the results shown that 7 of 10 were single copy and 3 were double copy. Furthermore, Southern blot and segregation ratio analysis were performed to validate this system, and when compared with traditional singlex qRT-PCR, this system improved the stability and accuracy of detection result. Therefore, this system could be great help for plant biotechnologists to fast,accurately and high-throughput identify endogenous gene copy number and homozygous plant.

关 键 词：转基因水稻 拷贝数 多重荧光定量PCR 

分 类 号：S184[农业科学—农业基础科学]