关键词: 柑橘, 实时荧光定量PCR, 转基因, 拷贝数, Southern blot

Abstract:

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.

Key words: citrus, quantitative real-time PCR, transgene, copy number, Southern blot