基于实时荧光定量PCR对转基因樱桃番茄外源基因拷贝数的检测

Detecting Exogenous Gene Copy Numbers of Exogenous Gene in Transgenic Tomato Based on Fluorescent Quantitative Real-time PCR

转基因植物中外源基因的整合拷贝数是影响外源基因表达水平及遗传稳定性的重要因素之一。本研究利用实时荧光定量PCR技术并以番茄抗坏血酸过氧化物酶apx（ascorbate peroxidase）基因和光诱导的早期蛋白elip（early light inducible protein）基因为内参开展对12株转基因樱桃番茄外源基因拷贝数的检测。研究结果表明：常规番茄使用的内参基因apx在樱桃番茄中可能以多拷贝形式存在;以elip基因为内参检测到转基因植株内整合的拷贝数为0~20不等,其中4株在普通PCR检测中存在假阳性,且80%的转基因植株发生了基因重排现象。

The copy number of foreign gene is an important factor that can greatly influence the level of expression and genetic stability in transgene plants.Early light-inducible protein（elip） and ascorbate peroxidase（apx）gene in tomato（Solanum lycopersicum L.） were chosen as endogenous gene and real-time fluorescent quantitative PCR were used to analyze the copy number in 12 transgenic lines in this research.Results showed that apx gene had more than one copy number in S.lycopersicum L.var.cerasiforme,there were 0 to 20 copy numbers using elip endogenous gene among 12 lines,and four of them were false positive in detection of conventional PCR.By comparing the copy number of each line using different exogenous gene,the number of integrated copies of the two genes was different and rearrangements occurred in eighty percent of 12 lines.