小菜蛾Treh基因启动子功能分析

Trehalase (Treh), which play an important physiological function in insect chitin synthesis and energy metabolism and combat environmental stresses, can hydrolysis trehalose into glucose. Interfering Treh gene expression to break the metabolic balance in insects it is an effective way for pest control. Now, expression and regulation of Treh are not clear. In this study, Plutella xylostella will be used as material, the function of PxTreh promoter by molecular biology and bioinformatics technology will be analyzed and the PxTreh promoter sequence of Plutella xylostella will be achieved. Next, dual luciferase reporter assay system will be constructed to identify PxTreh gene promoter activity and the important regulatory regions will be identified by the truncated 5 'and 3' end sequence. According to specific sequence in 5' upstream of PxTreh gene transcription starting point, transcription factors will be analyzed which has the regulation function using pull down technology and LC/MS mass spectrometry. Last, we will inhibit the expression of these factors to study the influence on growth and reproduction of plutella xylostella. The results of the study will be helpful to clarify expression and regulation mechanism of Treh in Plutella xylostella and be useful for designing new pesticides based on insect Treh gene transcription factors.

海藻糖酶可以将海藻糖水解成葡萄糖，在昆虫能量代谢、几丁质合成和抗逆等方面发挥重要生理功能。干扰昆虫海藻糖酶基因表达，可以打破昆虫体内的代谢平衡，是害虫防治的有效策略。但目前对Treh基因的表达调控机制尚未明确。本研究拟以小菜蛾为供试材料，采用分子生物学技术扩增获得小菜蛾Treh基因启动子序列；构建双荧光素酶报告基因检测系统鉴定PxTreh基因启动子活性，通过5'和3'端序列截短鉴定重要调控区域；根据PxTreh基因特异性的调控序列，使用pull-down技术和LC/MS质谱分析具有调控作用的转录因子，抑制具有重要调节作用的转录因子表达，研究其对小菜蛾生长发育和繁殖的影响。研究结果有利于阐明小菜蛾Treh基因表达调控机制，也可为以昆虫Treh基因转录因子为靶标设计新型杀虫剂提供科学依据。

海藻糖酶（Trehalase，Tre）可以将海藻糖水解成葡萄糖，是昆虫能量代谢必不可少的一类酶，也是昆虫体内几丁质合成途径的第一个酶。干扰海藻糖酶基因表达，是极具潜力的害虫防治策略。但目前对该类基因的表达调控机制尚未明确。本研究以小菜蛾为研究对象，利用去帽法5' RACE扩增了PxTre1和PxTre2起始密码子上游的转录本序列，明确了二者的转录起始位点，进一步通过基因组序列比对和5'侧翼序列扩增，获得了PxTre1和PxTre2转录起始位点之前1.5 kb以上的启动子序列。通过对两个基因启动子先后进行两轮截短，结合双荧光素酶报告基因检测系统，在sf9昆虫细胞中检测了不同长度启动子的活性，发现PxTre1启动子-487 bp至-426 bp存在转录正调控元件，-426 bp至-377 bp存在负调控元件；PxTre2启动子-203 bp至-90 bp对转录活性的影响至关重要。启动子关键区段的转录因子预测发现，CEBPα等转录因子可能调控了PxTre1和PxTre2的表达。本研究的结果有利于阐明小菜蛾Treh基因表达调控机制，也将为以昆虫Treh基因转录因子为靶标设计新型杀虫剂提供科学依据。