家蚕中肠特异启动子BmAPN的克隆及活性分析

【目的】克隆和鉴定1个家蚕中肠特异启动子，为研究家蚕的免疫应答和应用研究提供新的工具。【方法】从家蚕品种大造中提取基因组总DNA，用PCR方法克隆家蚕中肠特异表达基因BmAPN（GenBank登录号：BAA33715.1）的上游调控序列。利用此序列构建以EGFP为报告基因的转基因表达载体pBac[BmAPN-EGFP-SV40，3×P3-DsRed]，通过显微注射获得转基因家蚕，在个体水平上检测启动子的活性。【结果】所克隆的BmAPN启动子长度为1 598 bp，其驱动的EGFP仅在转基因家蚕中肠特异表达，与内源基因BmAPN的表达特征一致。该启动子在家蚕幼虫时期活性相对较高，且在起蚕期的活性要明显高于眠蚕期，推测该启动子中的特异元件受激素调控。【结论】经克隆获得的BmAPN启动子为有活性的启动子，且为家蚕中肠特异启动子。

Cloning and Activity Analysis of a Midgut-Specific Promoter BmAPN in Silkworm (Bombyx mori)

【Objective】The objective of this study is to provide a new tool for the immune-response and application research, and to clone and identify a midgut-specific promoter from Bombyx mori.【Method】 The 5′ upstream regulatory sequence of midgut-specific gene BmAPN (GenBank accession: BAA33715.1) was obtained by PCR from B. mori genomic DNA. A transgenic expression vector named pBac[BmAPN-EGFP-SV40, 3×P3-DsRed] was constructed, in which the reporter gene EGFP was driven by the BmAPN promoter. Furthermore, transgenic B. mori lines was obtained by microinjection and the activity of the promoter at the individual level were detected.【Result】The EGFP driven by a 1 598 bp BmAPN promoter was just specifically expressed in B. mori midgut. It was consistent with the expression pattern of endogenous gene BmAPN. The promoter activity was higher in B. mori larvae than other stages. And the activity in newly exuviated larvae was higher than that of molting larvae. It was speculated that specific components of the promoter might be regulated by hormone.【Conclusion】The BmAPN promoter is an active and midgut-specific promoter in B. mori.