棉铃虫胆固醇载体蛋白-2基因启动子的克隆与功能分析

The cotton bollworm, Helicoverpa armigera, is a major agricultural pest throughout the world. Helicoverpa armigera developed a strong resistance to many insecticides. The exploring of insecticides with new mode of action has been received increasing attentions. The sterol carrier protein-2 (SCP-2) was proved to be involved in intracellular cholesterol transfer in insects. It will be a potential new target for controlling the pests by affecting their cholesterol metabolism process. The Helicoverpa armigera sterol carrier protein-2 gene was first identified and proved to be important for normal development and fertility of Helicoverpa armigera. But the regulation of Helicoverpa armigera SCP-2 gene expression and transcription is not clear yet. In this project, we plan to clone the promoter of Helicoverpa armigera SCP-2 gene by chromosome walking. By transient transfection of promoter/reporter plasmids into lepidopteran insect cells, the promoter activities will be studied. Then, using the bioinformatics software to analyze and speculate the promoter structure. By constructions of a serial deleted promoter/reporter plasmids and transfection of insect cells, the activities of different promoter regions will be studied and the important spatial/temporal regulatory motif will be identified. All the results of these studies will lay an important basis for investigating the mechanism of Helicoverpa armigera SCP-2 expression and transcription regulation. This study will facilitate the screening of effective environment-friendly insecticides targeting SCP-2 and exploring the new way of pest control by targeting the insect cholesterol nutrition metabolism.

棉铃虫是抗药性很强的世界性农业主要害虫，研发对这类害虫有独特作用机理的杀虫剂备受关注。胆固醇载体蛋白-2(SCP-2)基于其运输胆固醇的重要作用，被证明是一种能通过影响虫体胆固醇代谢来防治害虫的潜在新型分子靶标。本实验室首次克隆了棉铃虫SCP-2基因，并证明了其在棉铃虫生长发育中的重要生理功能，但该基因的表达与转录调控目前尚不清楚。本项目拟开展棉铃虫SCP-2基因转录调控的重要元件-启动子的研究，采用染色体步移法克隆该基因启动子，通过鳞翅目昆虫细胞瞬时转染和报告基因系统检测启动子活性，利用生物信息学方法分析和预测启动子结构，构建一系列缺失的启动子/报告基因载体，比较启动子不同区域的活性，鉴定棉铃虫SCP-2启动子中重要的功能区域。本研究将为揭示棉铃虫SCP-2的表达与转录调控机理奠定重要的基础，为研发以SCP-2为靶标的绿色杀虫剂，靶向抑制昆虫营养代谢的害虫防治新途径提供理论依据。

棉铃虫是世界性农业主要害虫，杀虫剂的长期使用导致棉铃虫产生较强的抗药性和Bt抗虫棉耐受性，因此迫切需要开发安全高效的生物杀虫剂。胆固醇运输蛋白2（SCP-2）是昆虫体内一种重要的非特异性固醇运输蛋白，是开发新型杀虫剂的优良潜在靶点。本实验室首次克隆了棉铃虫SCP-2（HaSCP-2）基因并证明了其重要功能，本项目在此基础之上，按计划开展了棉铃虫SCP-2基因转录调控的重要元件-启动子的研究，克隆和分析了调控棉铃虫SCP-2基因表达的启动子，在体外培养的细胞中检测了启动子活性，并鉴定了调控基因表达的重要区域。取得了以下重要研究结果：（1）通过染色体步移，成功克隆了棉铃虫SCP-2基因ORF区上游约8kb侧翼序列；（2）构建了一系列截短启动子/荧光素酶报告基因重组质粒，采用双荧光素酶报告基因检测系统测定了启动子不同区域的活性，结果表明其中位于3’端1.8kb区域启动子最高，推测该区域对调控HaSCP-2基因表达非常重要；（3）通过5’-RACE分析，发现棉铃虫SCP-2基因启动子区存在多个转录起始位点，转录调控较为复杂；（4）对HaSCP-2启动子的生物信息学分析发现，该转录起始位点上游37nt区域可能存在CEBP、AP-1、TGIF等顺式作用元件, 这些元件的突变造成启动子活性显著降低，推测其为调控SCP-2表达的重要元件。本研究为揭示棉铃虫SCP-2的表达与转录调控机理打下重要的基础，为利用该潜在靶位点开发新型高效杀虫剂提供了重要理论依据，结合本课题组基于NMR技术首次获得的HaSCP-2蛋白的三维结构，将为研发SCP-2靶标杀虫剂，达到通过抑制昆虫营养代谢来防治害虫的目标而奠定重要理论基础。