反向PCR法扩增花粉特异表达基因PSG076启动子

Isolation of the Promoter Region of a Pollen-specific Gene PSG076 by Inverse-PCR

PSG076基因是从奥地利小麦品种‘Ferdinand’中分离的花粉特异性表达基因,功能未知。为获得可用于小麦基因工程的花粉特异性启动子,采用优化的反向PCR法分离PSG076启动子,获得了起始密码子上游约1.4 kb的启动子序列。生物信息学分析显示,该启动子除含有与花粉特异性表达相关的调控元件AGAAA和GTGA外,还含有花粉特异性表达相关的数量元件AGGTCA和AAATGA,推测其为活性较强的花粉特异性启动子。实验中对反向PCR方法的优化可提高扩增侧翼未知序列的效率,特别适用于启动子序列的扩增。

A pollen-specific gene,PSG076,with unknown function,was isolated from Austrian wheat ’Ferdinand’.To obtain pollen-specific promoter in wheat genetic engineering,the promoter region of PSG076 gene was isolated by an improved inverse-PCR（IPCR） method.A 1.4 kb promoter sequence was cloned and analyzed by Bioinformatics tools.Some cis-acting elements or enhancers conferred to pollen-specific expression were found,such as AGAAA,GTGA,AGGTCA and AAATGA.This suggested that PSG076 promoter might be a pollen-specific promoter with strong activity.The results also showed that the improved IPCR method was efficient to isolate the flanking unknown sequence,especially in promoter isolation.