美洲棉铃虫CYP321 A1基因启动子区萤火虫荧光素酶报告基因载体的构建及活性测定

Construction and functional identification of the luciferase reporter vectors of CYP321 A1 gene promoter

【目的】构建美洲棉铃虫细胞色素P450基因CYP321 A1启动子区萤火虫荧光素酶报告基因载体,为探索CYP321 A1基因的转录调控机制奠定基础。【方法】设计合成PCR引物,从美洲棉铃虫基因组DNA中扩增并克隆CYP321 A1基因的启动子区;将克隆的启动子片段插入载体pGL3-basic构建萤火虫荧光素酶报告基因载体p(-1 470/+64);用p(-1 470/+64)转染美洲棉铃虫脂肪体细胞系BCIRL-HzFB33,并用双荧光素酶报告基因检测系统分析该启动子活性。【结果】p(-1 470/+64)经双酶切、PCR鉴定及DNA测序分析鉴定准确无误;通过转染细胞和荧光素酶活性分析,证实所构建的重组载体p(-1 470/+64)可以反映CYP321 A1启动子活性;黄酮、花椒毒素处理极显著地增强了重组载体p(-1 470/+64)的荧光活性。【结论】成功构建了美洲棉铃虫细胞色素P450基因CYP321 A1启动子区萤火虫荧光素酶报告基因载体,CYP321A1启动子活性可以被植物次生物质黄酮、花椒毒素高度诱导。

【Objective】 The research was conducted to construct a luciferase reporter gene plasmid driven by CYP321 A1 gene promoter from Helicoverpa zea.【Method】 The promoter of CYP321 A1 gene was obtained by PCR amplification and was inserted into luciferase reporter gene plasmid pGL3-basic,the recombinant was identified by restriction enzyme digestion,PCR and DNA sequencing.The recombinant plasmid p（-1 470/＋64） was transiently transfected into H.zea fatbody cells,and promoter activity was measured with Dual-Luciferase Reporter Assay System.【Result】 The plasmid was constructed correctly as verified by double enzyme digestion,PCR identification and sequence analysis.Luciferase activity assay indicated that luciferase reporter plasmid p（-1 470/＋64） had significant luciferase activity;the expression level of p（-1 470/＋64） increased obviously after treatmemt by xanthotoxin or flavone.【Conclusion】 A luciferase reporter gene plasmid driven by CYP321 A1 gene promoter has been constructed successfully,the promoter activity of CYP321 A1 gene can be highly induced by plant allelochemical flavone or xanthotoxin.The research would provide a foundation for the study of the transcription regulatory mechanism of CYP321A1 gene.