AcMNPV polh启动子上游增强序列的部分缺失分析

本实验构建了2个用于杆状病毒AcMNPV(苜蓿丫纹夜蛾核多角体病毒)启动子及其调控序列研究的质粒载体:pF SBCAT和pF SBCATPpolh.以此为基础,结合目前对BmNPV(家蚕核多角体病毒)polh(多角体)基因和AcMNPVpolh基因上游增强序列的研究结果,构建了AcMNPVpolh基因上游增强序列部分缺失的瞬时表达载体;以cat为报告基因,利用瞬时转染和CATELISA的方法检测报告基因的表达活性,初步研究了对于polh启动子具有增强作用的AcMNPVpolh基因上游序列的结构.结果显示,与BmNPVpolh上游293bp序列同源性极高的AcMNPVpolh上游293bp片段对于polh启动子没有增强活性;CMVm启动子的AcMNPVpolh上游增强序列部分缺失之后不能够增强AcMNPVpolh启动子.因此,这一序列可能不再被缩短,也不大可能存在独立地起作用和更短的正、负调控元件.

Analysis on Partial Deletion of Upstream Enhancing Sequence of AcMNPV Polh Promoter

Two plasmid vectors: pF-SBCAT and pF-SBCATP_(polh) that can be used in researches of AcMNPV promoters and their regulating sequences were constructed. Basing on the research results of the pu (polh upstream) enhancing sequences of BmNPV and AcMNPV, a series of the pu enhancing sequences of AcMNPV that were deleted partially were inserted to AcMNPV polh promoter upstream of pF-SBCATP_(polh) separately;and when taking cat as reporter gene,by transient assay and CAT ELISA assay, the structure of the pu enhancing sequence of AcMNPV was inspected. Results show that the 293 bp pu fragment of AcMNPV which has highly identity with the 293 bp pu fragment of BmNPV can not stimulate AcMNPV polh promoter. When the pu sequence of AcMNPV that enhances CMVm promoter expression was partially deleted, it could not stimulate AcMNPV polh promoter. So, in order to keep it~,s enhancing function, the pu sequence might not be deleted. It is almost impossible that there is any shorter regulating element in the pu enhancing sequence.