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

发布时间：2018-06-08 16:35

本文选题：荧光定量PCR + 转基因绒山羊　；参考：《中国兽医学报》2017年08期

【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。
[Abstract]:Copy number of foreign genes is an important part of transgenic animal detection. In order to study the integration of exogenous genes of T 尾 4-GFP-FGF5s-GFP-VEGF164-GFP, three transtransposon systems of Piggy Bacillus were used to study the integration of exogenous genes in Northern Shaanxi White Cashmere Goats. The integrated copy number of exogenous gene copGFP was detected by fluorescence quantitative PCR. Using Gluc as internal reference gene, the standard curve equation of exogenous gene and internal reference gene was established. The DNA of transgenic Shanbei white cashmere goat was quantified by fluorescence PCR.The copGFP / Gluc ratio was used as the copy number of exogenous gene. At the same time, T 尾 4-GFP transgenic cashmere goats were selected to detect their integration sites to verify the results of copy number. The results showed that the standard curve equations for the two transgenic cashmere goats were: yyyyun-3.2306x 39.216kW ~ (20.998) -3.564 8x 38.440R2O0.9600.The number of integrative copies of 9 transgenic cashmere goats was identical with that of T 尾 _ 4-GFP transgenic cashmere goats by detection of the number of integrative sites and the number of copies of the transgenic Cashmere Goats (T 尾 _ 4-GFP). The results showed that the number of integrative sites in 9 transgenic Cashmere Goats was identical with that of Gluc goats. The results showed that FQ-PCR was a simple, sensitive and convenient method for detecting the integrated copy number of exogenous genes in transgenic cashmere goats.
【作者单位】：西北农林科技大学动物科技学院;榆林学院生命科学研究中心;陕西省陕北白绒山羊工程技术研究中心;
【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)
【分类号】：S827

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