斜纹夜蛾气味结合蛋白基因GOBP2转录调控区的克隆及活性分析  

Cloning and activity analysis of the transcription regulation region of GOBP2 in Spodoptera litura

作　　者：韩琪 胡波[1] 李升云 董双林[1] HAN Qi;HU Bo;LI Shengyun;DONG Shuanglin(College of Plant Protection,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学植物保护学院

出　　处：《南京农业大学学报》2019年第6期1059-1066,共8页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31672350,31872300)

摘　　要：[目的]本文旨在鉴定重要农业害虫斜纹夜蛾(Spodoptera litura)气味结合蛋白基因GOBP2的顺式作用元件,为揭示GOBP2的转录调控机制和开发基于嗅觉干扰的害虫防治技术奠定基础。[方法]利用5′RACE技术克隆GOBP2的5′UTR,确定转录起始位点;利用染色体步移技术(chromosome walking)克隆GOBP2的5′侧翼区,并利用在线网站预测转录因子结合位点;利用PCR克隆GOBP2的5′侧翼区逐步缺失片段,并构建到pGL3-Basic载体上;将各重组载体分别转染Sf9细胞,48 h后收获细胞并利用双荧光素酶报告系统检测转录活性。[结果]获得斜纹夜蛾GOBP2长23 bp的5′UTR,由此确定GOBP2的转录起始位点。通过染色体步移获得斜纹夜蛾GOBP2的5′端调控区2 025 bp的序列,发现启动子的典型TATA框;针对-1 000 bp序列的转录活性分析表明,最强转录活性区域位于-243^-207 bp,具有1个增强子;此外,在-971^-891 bp及-890^-811 bp各有1个增强子,在-244^-315 bp存在1个沉默子。[结论]获得斜纹夜蛾GOBP2的转录起始位点及顺式作用元件,为进一步研究GOBP2的转录调控机制奠定了基础。[Objectives]The purpose of this research is to identify the cis-elements of GOBP2 in the important agricultural pest Spodoptera litura,and to provide bases for clarification of the transcriptional regulation of GOBP2 and develop pest control techniques based on olfactory interference. [Methods]The 5′RACE was used to clone the 5′UTR,and the transcription start site was determined based on the 5′UTR. The chromosome walking was used to clone the 5′flanking region of GOBP2,and the transcription factor binding sites were predicted by online tools;PCR was used to clone the 5′-end progressive deletion sequences of the GOBP2,and pGL3-Basic vector was used to construct the recombinant vectors containing the deletion sequences;Sf9 cells were transfected with recombinant pGL3-Basic vector;the dual-luciferase reporter assay system was used to measure the transcriptional activity with cells harvested at 48 h post transfection. [Results]The 5′UTR of GOBP2 in 23 bp was verified,and thus the transcription start site was determined. The 5′ flanking region of 2 025 bp length of GOBP2 was obtained,and the TATA box of the promoter was identified. The dual-luciferase reporter gene assay showed that the strongest enhancer of GOBP2 was located in the 37 bp sequence from-243 bp to-207 bp. In addition,two enhancers and a silencer were indicated in-971 bp to-891 bp,-890 bp to-811 bp,and-244 bp to-315 bp,respectively. [Conclusions]The transcription start site and 4 cis-elements were obtained,providing bases for further clarifying the transcriptional regulation mechanism of GOBP2 in S.litura.

关 键 词：斜纹夜蛾 气味结合蛋白 双荧光素酶报告基因检测 启动子 增强子 沉默子 

分 类 号：S436.3[农业科学—农业昆虫与害虫防治]