家蚕卵黄原蛋白(BmVg)基因启动子的克隆与活性鉴定

Promoter Cloning and Activity Determination of Vitellogenin Gene in Silkworm,Bombyx mori

卵黄原蛋白(vitellogenin,Vg)在家蚕蛹期脂肪体高量表达,研究家蚕卵黄原蛋白基因(BmVg)的转录调控机制及启动子活性,将有助于利用家蚕蛹生物反应器高效表达外源蛋白。设计特异引物,以家蚕蛹基因组DNA为模板PCR扩增得到BmVg长约1.5 kb的启动子片段。构建由不同长度截短BmVg启动子片段驱动荧光素酶报告基因表达的细胞转染载体pGL3-Vg Promoter,分析转染家蚕胚胎培养细胞BmE-SWU1中的BmVg启动子片段活性,结果显示最低截短至BmVg基因翻译起始位点上游90 bp的启动子片段具有转录活性。进一步用截短法分析不同长度启动子的活性,当启动子-752^-952 bp、-42^-152 bp区域被截掉后,启动子的转录活性明显降低,暗示这2个区域内可能存在与启动子启动效率相关的重要调控元件。

Vitellogenin is highly expressed in the fat body of silkworm （Bombyx mori） pupa. The study of transcriptional regulation mechanism of Bombyx mori vitellogenin （BmVg） gene and its promoter activity can help us to express exoge- nous proteins efficiently using silkworm pupae as bioreactor. In this study, 1.5 kb BmVg promoter fragment was amplified by PCR from the silkworm pupal genome DNA. The cell transfection vector named pGL3-Vg Promoter was constructed using differently truncated BmVg promoter to drive luciferase gene. Then, the BmVg promoter activity was analyzed in the BmE-SWU1 cell line. The results showed the BmVg promoter with 90 bp from the translation initiation site had basic tran- scription activity. Furthermore, the promoter activity was reduced obviously when the area -752 to -952 bp and -42 to -152 bp was deleted, which implied that there are important elements to the promoter activity in these two areas of BmVg promoter.