利用多重荧光定量PCR检测转基因水稻外源基因拷贝数

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,hptII)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,OsSFu2a)作为内参基因,构建基于Taqman的多重荧光定量PCR(Multiplex qRT-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hptII-1和OsSFu2a-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Southem 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 qRT-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 (hptII),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 hptII-1 and OsFU2a-2 was the best one.The copy 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,Southem 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.