大豆中疫霉诱导性GmDRRP基因启动子的克隆与功能分析

Identification and functional characterization of the Phytophthora sojae induced-promoter of the soybean GmDRRP gene

本研究根据大豆与疫霉互作的基因芯片数据,筛选了一个受疫霉诱导表达的大豆抗病相关基因(Gm DRRP,glycine max disease resistance response protein),并对其启动子响应疫霉侵染的功能进行了研究。序列分析表明该基因编码一个大豆抗病相关蛋白。分别用SA、Me JA、ABA、ETH和GA3五种激素处理后,发现该基因的表达受激素的抑制。克隆其转录起始位点上游1 590 bp的启动子区,生物信息学分析发现该区域包含多个已知与逆境响应相关的顺式元件。进一步将启动子构建在融合Gus报告基因的植物表达载体上,分别瞬时转化烟草和稳定转化大豆根毛,并检测了Gus报告基因的表达情况。结果表明,Gm DRRP启动子均能在两种体系中不同程度的受疫霉诱导,其表达模式为接种后0.5 h被快速诱导,并在2 h时显著提高。根据生物信息学对顺式元件的预测结果,对启动子进行了分段分析,获得了一个222 bp的小片段,其疫霉诱导表达能力是全长启动子的34%。以上结果表明大豆的Gm DRRP启动子能被疫霉快速诱导。

In this study, we obtained a GrnDRRP （glycine max disease resistance response protein） gene from soybean microarray data, and functionally characterized roles of its promoter during Phytophthora sojae infection. Phylogenetic and functional domain analysis showed that this gene encoded a soybean DRRP protein. qRT-PCR analysis indicated it was down-regulated by the tested hormones. Then, a promoter fragment harboring the -1,590 to ＋1 region （the transcription start site of GmDRRP is defined as ＋1 position） was isolated from soybean （cv. Williams）. Its activity was determined by both transient expression assay in Nicotiana benthamiana and stable expression in transgenic soybean hairy roots. The results showed that GmDRRP promoter-derived Gus expression was rapidly induced at 0.5 hpi （hours post infection）, and reached high levels at 2 hpi. We also identified a 222 bp fragment that was important for its full activities by deletion analysis. Thus, we provide a promoter that can confer Phytophthora-induced expression.