一个黑曲霉细胞色素P450基因启动子的结构与表征

Structure and Characterization of the Promoter of Aspergillus niger Cytochrome P450 Gene

黑曲霉是工业生产上的重要模式菌株,其启动子活性对其生产效率有重要影响;细胞色素P450是调控菌体能量代谢、细胞生长和各种代谢产物合成的关键酶.通过对细胞色素P450基因启动子区域(PP450)的克隆、结构分析和功能鉴定,为P450基因的表达调节研究和P450启动子的应用提供基础.从黑曲霉基因组克隆出细胞色素P450基因(An11g01980)的上游片段PP450,连接到pGEM-T载体进行测序,用启动子分析网站GPMiner和EMBOSS分析PP450片段的启动子功能元件,结果显示:PP450片段的-864^-106 bp处有一段长为759 bp的CpG区域,含有2个TATA盒、6个CAAT盒、2个GC盒和1个可能的AmyR结合位点.构建含PP450启动子的启动子–报告基因表达载体pPZP-PP450并转化黑曲霉,通过荧光强度检测启动子功能,结果表明该启动子是一个组成型启动子,但添加沃氏氧化物后启动子表达强度明显增强,推测该启动子区域可能含有沃氏氧化物相关的反应元件或增强子.

Aspergillus niger is an important model strain in industrial production,and its promoter activity has an important effect on its production efficiency.Cytochrome P450 is a key enzyme that regulates energy metabolism,cell growth and the synthesis of various metabolites.Through cloning,structural analysis and functional identification of the cytochrome P450 promoter region(PP450),the foundation for research on the expression regulation of P450 gene and the application of P450 promoter have been laid.PP450,an upstream segment of the cytochrome P450 gene(An11g01980),was cloned from A.niger genome,ligated into pGEM-T plasmid and sequenced to analyze its functional elements in PP450 using the promoter sites GPMiner and EMBOSS.The results show that PP450 has a 759 bp long CpG area between-864 and-106 bp,contains 2 TATA boxes,6 CAAT boxes,2 GC boxes and a possible AmyR binding site.The promoter-reporter expression vector pPZP-PP450 containing PP450 was thus constructed and then transformed in A.niger,and the function of PP450 was identified in fluorescence intensity test.The results show that P450 promoter is a constitutive promoter.However,the expression strength of P450 promoter was significantly enhanced after adding 16,17-α-epoxyprogesterone in culture.It is speculated that P450 promoter region may contain 16,17-α-epoxyprogesterone-related response elements or enhancer.