粘虫中肠胰蛋白酶基因启动子的克隆及其功能分析

Cloning and functional analysis of trypsin promoter from midgut of Mythimna separata (Walker)

【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。

[Objective] The 5p flanking promoter sequence of trypsin gene was cloned from midgut of Mythimna separate and their functions were studied to provide basis for further exploration of activity reg- ulatory mechanism of insect trypsin. [Method] The 5p flanking promoter sequence of trypsin gene was cloned from midgut of M. separata by Genome Walking. The sequence analysis was carried out by online software NNPP v. 2.2 and database JASPAR. Firefly luciferase reporter gene vector driven by trypsin gene promoter was built and transfected to Spodoptera frugiperda （J. E. Smith） （Lepidoptera：Noctuidae） sf21 ceils line. Then,the promoter activity was analyzed after transient expression by a dual-luciferase reporter assay system. [Result] The complete 5rflanking promoter sequence of trypsin gene was 1 863 bp containing TATA box, CAAT box and other core promoter sequences. GATA, STAT, C/EBP and other transcription- al regulatory elements were also contained in this sequence. The promoter activity in the recombinant vec- tor p（--1 673/＋25） was significantly higher than that in pGL3-Basic vector by the analysis of Dual-Lucif- erase reporter assay system. [Conclusion] The cloned promoter fragment was able to promote the high ex- pression of firefly luciferase reporter gene and could be used to study the activation mechanism of periplo-cosides compounds in the level of trypsin gene promoter and transcription factors.