粘虫中肠胰蛋白酶基因启动子的克隆及其功能分析

发布时间：2018-04-04 19:48

本文选题：粘虫　切入点：胰蛋白酶　出处：《西北农林科技大学学报(自然科学版)》2017年06期

【摘要】：【目的】通过克隆粘虫Mythimna separata(Walker)(Lepidoptera:Noctuidae)中肠胰蛋白酶基因5′端侧翼启动子序列,并分析启动子功能,为进一步探索昆虫胰蛋白酶活性调控机制奠定基础。【方法】利用Genome Walking方法克隆粘虫中肠胰蛋白酶基因5′端侧翼启动子序列,运用在线分析软件NNPP v.2.2和数据库JASPAR进行序列分析,构建由胰蛋白酶基因启动子驱动的萤火虫荧光素酶报告基因载体,通过转染草地贪夜蛾sf21细胞系,瞬时表达后用双荧光素酶报告基因检测系统分析该启动子活性。【结果】克隆得到粘虫中肠胰蛋白酶基因5′端侧翼启动子序列1 863bp,其中含TATA框、CAAT框等启动子核心序列及GATA、STAT、C/EBP等转录调控元件。双荧光素酶报告基因检测系统分析表明,相对于空载体pGL3-Basic,所构建的重组载体p(-1 673/+25)具有明显的启动子活性。【结论】克隆得到的启动子片段明显具有启动报告基因表达的能力,可用于在胰蛋白酶基因启动子水平和转录因子水平研究杠柳新苷活性化合物的激活机理。
[Abstract]:[objective] to clone the 5 'flanking promoter sequence of the midgut trypsin gene of Mythimna separata Walkeria: Noctuidae, and to analyze the promoter function.[methods] Genome Walking method was used to clone the 5 'flanking promoter sequence of intestinal trypsin gene.Using online analysis software NNPP v.2.2 and database JASPAR for sequence analysis, a fluorescent luciferase reporter gene vector driven by trypsin gene promoter was constructed and transfected into sf21 cell line.After transient expression, the promoter activity was analyzed by double luciferase reporter gene detection system. [results] the 5'flanking promoter sequence of 5 'trypsin gene from Myxomycetes was cloned into 1863 BP, which contained TATA frame, CAAT frame and other promoter nuclei.Cardiac sequence and transcriptional regulatory elements, such as GATAA STATX C / EBP.The analysis of double luciferase reporter gene system showed that the recombinant vector pGL-673 / 25 had obvious promoter activity compared with the empty vector pGL3-Basic.Conclusion the cloned promoter fragment has the ability to initiate reporter gene expression.It can be used to study the activation mechanism of the active compounds at the level of trypsin gene promoter and transcription factor.
【作者单位】：西北农林科技大学植物保护学院;西北农林科技大学林学院;
【基金】：国家重大基础研究规划(“973”计划)项目(2010CB126100) 国家自然科学基金项目(31071704,31300548) 陕西省农业科技攻关计划项目(2014k02-10-02)
【分类号】：Q966

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