利用荧光定量PCR法检测转基因绒山羊外源基因整合拷贝数  

Estimating the copy numbers of exogenous gene in transgenic cashmere goats by real-time fluorescence quantitative PCR

作　　者：师丙波 黄玉[1] 何晓琳[1] 朱海鲸[2,3] 于鸿浩[2,3] 金妙函 屈雷[2,3] 陈玉林[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]榆林学院生命科学研究中心,陕西榆林719000 [3]陕西省陕北白绒山羊工程技术研究中心,陕西榆林719000

出　　处：《中国兽医学报》2017年第8期1605-1612,共8页Chinese Journal of Veterinary Science

基　　金：高产绒量转基因绒山羊新品种培育专项资助项目(2014ZX08008-002);国家绒毛用羊产业技术体系(舍饲半舍饲营养岗位)资助项目(CARS-40);国家自然科学基金资助项目(31372279;31402038)

摘　　要：外源基因拷贝数是转基因动物检测中的重要内容。本实验室使用PiggyBac(PB)转座子系统获得Tβ4-GFP、FGF5s-GFP、VEGF164-GFP 3种转基因类型陕北白绒山羊,为研究其外源基因整合情况,利用荧光定量PCR方法对其外源基因copGFP的整合拷贝数进行检测。以Gluc为内参基因,建立外源基因和内参基因的标准曲线方程,对转基因陕北白绒山羊DNA进行荧光定量PCR,将copGFP/Gluc的比率作为外源基因的拷贝数。同时,选取Tβ4-GFP转基因绒山羊对其整合位点进行检测,以验证拷贝数结果。结果显示:copGFP与Gluc的标准曲线方程分别为,y=-3.230 6x+39.216(R^2=0.998 8)、y=-3.564 8x+38.440(R^2=0.996 0);成功得到9只转基因绒山羊的整合拷贝数;Tβ4-GFP转基因绒山羊整合位点数目和拷贝数检测结果一致。结果表明,利用荧光定量PCR检测转基因绒山羊外源基因整合拷贝数,操作简单、灵敏方便,是一种切实可行的检测方法。The copy numbers of exogenous gene in transgenic animals is always regarded as an important information of transgenic animals. Thus,simple and sensitive methods are required for the detection of the copy numbers of exogenous gene. Three kinds of transgenic Shanbei white cash- mere goats, containing Tβ4-GFP, FGFSs-GFP and VEGF164-GFP, has been obtained by using Pig- gyBac（PB） transposon system. Fluorescence quantitative PCR was carried out to detect the copy numbers of copGFP. Using Gluc as reference gene,the double standard curves of exogenous gene and reference gene were mapped and the genomic DNA of transgenic goats were analysized by real- time fluorescence quantitative PCR. Moreover,the copGFP/Gluc ratio in the samples was calculated as the copy numbers of copGFP. In addition,Tβ4-GFP transgenic cashmere goats were selected to detect the integration sites by using the genomic walking kit. The results showed that the stand- ard curve equation of copGFP was y=-3. 230 6x＋39. 216 （R^2=0. 998 8） and the standard curve equation Of Gluc was y=-3. 564 8x＋38.440 （R^2 =0. 996 0）. The copy numbers of exogenous gene in the transgenic cashmere goats were obtained and the numbers of integration sites in the selected Tβ4-GFP transgenic goats were consistent with the copy numbers of copGFP. As a conclu- sion,the high-throughput, fast and sensitive real-time fluorescence quantitative PCR is an efficient and convenient method for the copy number of exogenous gene in transgenic cashmere goats.

关 键 词：荧光定量PCR 转基因绒山羊 拷贝数 

分 类 号：S813.3[农业科学—畜牧学]