马铃薯损伤诱导型启动子Wun1基因的克隆及其GFP表达活性

通过聚合酶链式反应(PCR),以马铃薯基因组DNA为模板,根据已报道Wun1序列设计了一对特异引物,在优化的PCR反应条件下扩增出了Wun1基因片段,通过序列分析与文献报道的碱基序列有96.86%的同源性,该基因已登录到GenBank(No.AY803296)。以pBIPG(携带GFP基因)的质粒DNA为模板,通过PCR技术亚克隆到了源自水母(Aequorea)大小为756bp的绿色荧光蛋白(GFP)基因,与已知序列同源率为100%。利用GFP基因作为报告基因,构建了用于比较鉴定所克隆启动子活性的pBIG(35S-GFP)和pBIWG(Wun1-GFP)两个植物表达载体,采用基因枪法进行对洋葱表皮细胞的遗传转化,检测Wun1启动子在受体细胞中调控基因表达的活性,结果表明克隆到的Wun1启动子活性强于组成型表达的35S启动子,GFP瞬时表达的分析方法也让我们有效的筛选到用于进行马铃薯抗病育种的调控元件。

Cloning of Potato Wound-inducible Promoter Wun1 and its GFP Express Activity

In present studies, a 1 239 bp fragment of potato wound-inducible promoter Wun1 was cloned via polymerase chain reaction (PCR) using the genome DNA extracted from tissue-cultivated potato plant as template. Sequence analysis indicates the cloned fragment shares 96.86% homology with the reported sequence, the difference between them is also remarkable. As a new promoter it has been registered in GenBank (No. AY803296). Via PCR and using plasmid pBIPG DNA as template, the GFP gene which comes from Aequorea is cloned. Analysis indicates the homology between this clone and reported sequence is 100%. Then utilizes the gene of green fluorescence protein (GFP) as reporting gene, two transient expressed plant expression vector pBIG(35S-GFP) and pBIWG(Wun1-GFP) is constructed by combined the interest gene with the pBI121 vector, transform the epidermis cells of onion by particle bombardment to detect the Wun1 promoter which regulates gene expression activities among recipient cells. Result shows intensity in transformed pBIWG is higher than in transformed pBIG obviously under fluorescence microscopy.