用差异显示PCR技术克隆冬小麦春化设定cDNA克隆实验系统的建立

ESTABLISHMENT OF STUDYING SYSTEM CLONED VERNALIZATION-PUTATIVE cDNA CLONE BY MEANS OF DIFFERENTIAL DISPLAY PCR

差异显示PCR技术是一种新近发展起来的在真核细胞中检测与克隆特异表达基因的手段。本文建立了用此技术克隆春化相关基因的研究系统,并提供了诸如总RNA的提取、污染DNA的去除、sscDNA的逆转录、PCR参数、扩增cDNA的电泳、特异cDNA的收集与重扩增等在方法上的细节。用这种技术分离了一个仅在春化20天这一特异时期表达的春化设定cDNA克隆VPC28。这些结果也适用于更加广泛的类似研究。

Differential display PCR has been developed as a tool detecting and characterizing specially expressed gene in eukaryotic cells recently. In this paper, a studying system was established which applied the novel approach of differential display PCR to clone vernalization-related cDNA clone. Here methodological details were provided which included the purification of total RNA,removal of contaminated DNA, reverse transcrip- tion of cDNAs, parameters of PCR, elec-trophoresis of amplified cDNAs, recovery and reamplification of cDNAs. It was also identified a vernalization-putative cDNA clone (VPC ) of VPC28 which was only expressed at the key stage of vernalization for 20 d in winter wheat. These results moreover made it readily applicable to a broad spectrum of similar studies.