拟南芥 AtRBOHD 基因表达载体构建及鉴定分析

AtRBOHD 基因作为植物体内重要的 NADPH 氧化酶基因, 可将氧催化生成活性氧以响应逆境胁迫, 但其是否参与植物重金属镉胁迫的调控尚不清楚。文章以拟南芥为实验材料, 利用聚合酶链式反应(polymerase chain reaction, PCR)技术克隆出 AtRBOHD 基因的启动子区域, 成功构建 ProAtRBOHD:GUS 融合表达载体; 并通过浸花法将该融合表达载体转化到野生型拟南芥中, 通过抗性筛选、PCR 鉴定、抗性分离鉴定筛选出纯合阳性转基因植株。进一步对获得的转基因材料进行镉胁迫处理, 发现经处理后的转基因材料 GUS 活性提高。为初步研究镉胁迫下 AtRBOHD 基因功能和转录水平变化提供重要依据。

As an important NADPH oxidase gene in plants, AtRBOHD gene can generate reactive oxygen species by catalyzing oxygen in response to adversity stress, but whether it participates in the regulation of plant heavy metal cadmium stress is unclear. In this study, using Arabidopsis thaliana as experimental material, the promoter region of AtRBOHD gene was cloned by polymerase chain reaction (PCR) technology, and the ProAtRBOHD:GUS fusion expression vector was successfully constructed. The fusion expression vector was transformed into wild-type Arabidopsis thaliana by floral dip method, and homozygous and positive transgenic plants were screened out by resistance screening, PCR identification and resistance isolation identification. Furthermore, cadmium stress was performed on the obtained transgenic materials, and it was found that the GUS activity of the treated transgenic materials increased. This study provides an important basis for the preliminary research on the function and transcription level of AtRBOHD gene under cadmium stress.