结缕草胁迫诱导型启动子Rd29A的克隆及功能鉴定

Cloning and Identifying Stress-inducible Promoter Rd29A of Zoysia japonica Steud.

以结缕草基因组DNA为模板,根据已报道Rd29A启动子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Rd29A启动子的基因片段,通过序列分析与文献报道的基因序列有41.65%的同源性。在GenBank上的登录号为EU346948。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pB IG(35S-GFP)和pB IRG(Rd29A-GFP)两个植物表达载体,进而采用基因枪法对洋葱表皮细胞进行遗传转化,检测Rd29A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Rd29A启动子活性强于组成型的35S启动子,利用GFP瞬时表达的分析方法有效的筛选到用于进行草坪草抗逆育种的启动子。

A fragment of stress-inducible promoter Rd29A was cloned using the genome DNA extracted from tissue-cultivated Zoysia japonica Steud.plant as template.Sequence analysis indicated the cloned fragment shared 41.65% homology with the reported sequence,and the difference between them was also remarkable.The number of accession GenBank is EU346948.Then utilizes the gene of green fluorescence protein（GFP）as reporting gene,two transient plant expression vectors pBIG（35S-GFP）and pBIRG（Rd29A-GFP）were constructed,transform the epidermis cells of onion by particle bombardment to detect the Rd29A promoter which regulates gene expression activities among recipient cells.Results showed intensity in transformed pBIRG is higher than in transformed pBIG obviously under fluorescence microscopy.The analysis method of GFP instantaneous expressing is effective to select promoter in resist-adversity breeding of turfgrass.