家蚕核多角体病毒解旋酶基因启动子及增强子hr3功能分析  

Functional Analysis of Helicase Gene Promoter and Homologous Region 3 Enhancer in Bombyx mori Nuclear Polyhedrosis Virus

作　　者：肖庆利[1] 张志芳[1] 易咏竹[1] 何家禄[1] 吴祥甫[2] 

机构地区：[1]农业部家蚕生物技术重点开放实验室,中国农业科学院蚕业研究所,镇江212018 [2]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海200031

出　　处：《生物化学与生物物理学报》2001年第5期525-530,共6页

基　　金：国家自然科学基金资助项目 (No .39970 5 71)&&

摘　　要：从BmNPVZJ8株克隆了解旋酶 (helicase)基因ATG上游的 5 10bp启动子 ,序列分析发现 ,该启动子同时具有早期和晚期RNA转录起始位点。将起始密码突变为ATT后 ,引入萤火虫荧光素酶 (Luc)基因作瞬时表达分析。用表达质粒 pBmhel5 10luc转染Bm 5和Sf 2 1细胞 ,解旋酶基因启动子能被细胞的RNA聚合酶识别 ,具有早期启动子的特性 ,且病毒因子对hel5 10启动子具有反式激活作用。杆状病毒同源重复区 (hr)序列是病毒DNA复制起始点 ,又具有增强子功能。将BmNPVhr3序列克隆到hel5 10启动子的下游进行瞬时表达 ,结果表明 ,hr3可增强hel5 10启动子在昆虫细胞和家蚕幼虫中的转录活性分别达 70 0 0倍和 10 0The promoter of the helicase gene, including 510 bp upstream of ATG, was cloned and sequenced, and was found that it had both early and late RNA initiation sites. The initiation codon ATG was deleted by using point mutation. Luciferase gene, as a reporter gene, was fused with the promoter region to construct the plsmid pBmhel510 luc. When pBmhel510luc was transfected into Bm5 and Sf21 cell lines, the helicase gene promoter was recognized by cellular RNA polymerase and transactivated by viral factors. Baculovirus homologous regions (hrs) act as viral DNA replication start sites, which also have been shown to alter the rate of transcription for cislinked promoters. BmNPV hr3 was cloned into a downstream site of luc gene, to study the effect of this enhancer on hel510 promoter activity. The transient expression in transfected insect cell lines and silkworm larvae indicated thathr3 could enhance the transcriptional level of hel 510 promoter by about 7 000 and 1 000 fold, respectively.

关 键 词：家蚕核多角体病毒 解旋酶基因启动子 增强子 瞬时表达 

分 类 号：Q753[生物学—分子生物学]