实时定量PCR检测转基因绵羊拷贝数  

Real- time Quantitative PCR Was Used to Detect Transgenic Sheep Exogenous Gene Copy Number

作　　者：赵帅[1,2] 王立民[2] 王聪慧[1] 唐红[2] 张宾[2] 郭延华[2] 张译元[2] 赵兴旺 丁新平 张银国[1] 周平[2] 

机构地区：[1]石河子大学动物科技学院,新疆石河予832000 [2]新疆兵团绵羊繁育生物技术重点实验室,新疆石河子832000 [3]石河子石总场畜牧兽医服务中心,新疆石河子832011 [4]新疆西部牧业股份有限公司紫泥采种羊场,新疆石河子832000

出　　处：《新疆农业科学》2015年第8期1517-1521,共5页Xinjiang Agricultural Sciences

基　　金：高产超细毛转基因羊新品种培育(2014ZX08008-001);国家自然科学基金项目(31160227);新疆生产建设兵团院士资金专项(2012BB024,2014BB023);新疆农垦科学院引导计划(49YYD201209)

摘　　要：【目的】利用高通量、快速、灵敏的实时荧光定量PCR法,检测转类胰岛素样生长因子1-红色荧光蛋白基因(insulin-like growth factor 1-red fluorescent protein,IGF1-RFP)绵羊中外源基因的拷贝数。【方法】以甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)作为绵羊的内源参照基因,梯度稀释法。【结果】分别获得RFP和GAPDH基因的Ct值与起始模板数的相关性标准曲线,R2分别为0.999和0.999,相关性高。通过比较目的基因RFP和GAPDH起始模板数,得到外源基因在转基因绵羊中的拷贝数。编号0152、0216、0217、0218的转基因绵羊拷贝数分别为3、1、1、1。【结论】以IGF-I转基因细毛羊为研究对象,针对外源基因序列和内源参照基因序列设计特异性引物,利用实时荧光定量PCR法对IGF-I转基因细毛羊外源基因的拷贝数进行检测,并且准确检测外源基因的拷贝数。【Objective】High- throughput,fast and sensitive real- time fluorescence quantitative PCR was used to estimate the copy number of insulin- like growth factor 1- Red fluorescent protein gene（ IGF1-RFP） in the transgenic sheep. 【Method】The glyceraldehyde- 3- phosphate dehydrogenase（ GAPDH） in sheep 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 RFP and GAPDH gene were obtained,and the correlation coefficients were 0. 999 and 0. 999,respectively. The transgenic copy number was obtained bycomparing the initial template copy of RFP with that of GAPDH. 【Result】The copy numbers of transgenic sheep No. 0152,0216,0217 and 0218 were 3,1,1,and 1,respectively.【Conclusion】The IGF- I transgenic fine- wool sheep for the study,the specific primers were designed for exogenous sequence and endogenous reference gene sequence,real- time quantitative PCR was used to detect the exogenous gene copy number of IGF- I transgenic fine- wool sheep and the copy number of exogenous gene was determined accurately.

关 键 词：转基因绵羊 实时荧光定量PCR 拷贝数 

分 类 号：S852.16[农业科学—基础兽医学]