利用实时荧光定量PCR法检测转基因水稻外源基因拷贝数的研究  

Estimation of the Copy Number of Exogenous Gene in Transgenic Rice by Real-time Fluorescence Quantitative PCR

作　　者：王育花[1] 赵森[1] 陈芬[1] 肖国樱[1] 

机构地区：[1]中国科学院亚热带农业生态研究所

出　　处：《生命科学研究》2007年第4期301-305,共5页Life Science Research

基　　金：中国科学院知识创新工程重要方向项目(KZCX3-SW-434);湖南省杰出青年科学基金(03JJY1004);湖南省"十一五"重大科技专项(2006NK1001)

摘　　要：转基因植物中外源基因拷贝数是影响目的基因表达水平和遗传稳定性的重要因素,因此外源基因拷贝数的检测成为转基因研究的关键.利用高通量、快速、灵敏的SYBR GreenⅠ荧光定量实时PCR法,检测了转大麦烟酰胺合成酶基因(NAS1)水稻中外源基因拷贝数.以蔗糖磷酸合成酶基因(SPS)作为水稻的内源参照基因,通过梯度稀释法,分别获得了NAS1和SPS基因的Ct值与起始模板数的相关性标准曲线,相关系数分别为0.99976和0.99571,相关性高.通过目的基因NAS1和水稻内源参照基因SPS起始模板数的比较,获得了目的基因在转基因水稻中的拷贝数,在8株转基因株系中,1株为假阳性,1株拷贝数为1,3株拷贝数为2,其余3株拷贝数分别为3、4和7,而阴性对照拷贝数为0.这种方法快速、简便、准确,可以满足转基因育种工作中对后代优良株系的选择.In transgenic plants, the number of transgene copy is an important factor that can greatly influence the level of expression and genetic stability of the target gene. Estimating the transgene copy number becomes a significant step in transgenic research. High-throughput, fast and sensitive SYBR Green I real-time fluorescence quantitative PCR was used to estimate the copy number of nicotianamine synthase gene （NAS1） of barley in transgenic rice, and the sucrose phosphate synthase gene （SPS） in rice was used as endogenous reference gene. With a serial of dilutions, the standard curves of the threshold cycle （Ct） relative to the log of each initial template copy of NAS1 and SPS gene were obtained, and the correlation coefficients were 0.99976 and 0.99571, respectively. The transgenic copy number was obtained by comparing the initial template copy of NAS1 with that of SPS. Among the eight putative transgenic lines, one was non-transgenic plant, one had one copy number, three had two copies, and the other had three, four and seven copies, respectively. At the same time, the negative control had zero copy. The method of estimating copy number of exogenous gene by real-time fluorescence quantitative PCR was characterized as speediness, breeding. convenience and nicety, which can satisfy the demands of elite line selection in transgenic breeding.

关 键 词：SYBR Green Ⅰ实时荧光定量PCR 烟酰胺合成酶基因 转基因水稻 拷贝数 

分 类 号：Q503[生物学—生物化学]