转基因柑橘外源基因拷贝数的实时荧光定量PCR检测

为分析转基因柑橘中外源基因整合拷贝数，基于实时荧光定量PCR法，建立通用、准确、简便的转基因柑橘外源基因拷贝数检测体系，利用该体系成功检测了150个转基因柑橘单株系外源基因整合拷贝数。结果表明，转基因柑橘中外源基因整合最低拷贝数为1，最高达5个，其中1、2和 ≥ 3拷贝的单株数分别占总数的40.0%、18.0%和42.0%；采用Southern blot技术进行检测，结果基本一致，极少数单株经实时荧光定量PCR分析的拷贝数比Southern blot检测的数值高。说明本研究建立的实时荧光定量PCR方法检测转基因柑橘外源基因整合拷贝数更加准确可靠。拷贝数为1 ~ 2的转基因株系可作为将来开展转基因目标性状研究及转基因生物安全性评价有利用价值的试验材料。

Identification of the Copy Number of Exogenous Gene in Transgenic Citrus by Quantitative Real-time PCR

The copy number of exogenous gene is an important factor influencing the expression and genetic stability of the target gene. Currently，transgene copy numbers by Southern blot，which is laborious and time-consuming，requires relatively large amounts of plant materials and more experimental technology. Here，based on quantitative real-time PCR（qRT-PCR）technology，a protocol for estimating transgene copy number in GM citrus was constructed. The results showed that the low copy number of exogenous gene in transgenic line was one，and the highest copy number was five. One，two and ≥ three copies detected accounted for 40.0%，18.0%，42.0%，respectively，of the total. The exogenous gene copy in the transgenic lines was further confirmed by Southern blot，and the copy number was consistent with the above two methods，although the copy number by qRT-PCR analysis in a few of plants was more than that by Southern blot. Therefore，the evaluation result with Real-time fluorescent quantitative PCR method was more accurate and reliable in comparison to that with Southern blot. And transgenic lines with a copy number of 1–2 could be used as an experimental material for the evaluation of the GMO bio-safety.