Functional Analysis Of The Cathepsin D Gene Promoter Of Bombyx Mori

Bombyx mori Cathepsin D (BmCatD) plays a very important role in regulatingmetamorphosis processis in silkworm. To better understand the regulation of molecularmechanisms, we conducted this study to identify the function of BmCatD promoter. In thisstudy, silkworm strains which are permissive to rAcMNPV were selected and used. Therecombinant AcMNPV (Autographa californica multiple nucleopolyhedrovirus) vector wasused as the transfer and luciferase assay with a dual-luciferase quantitative assay system wasused as the reporter gene to investigate the expression profiles of the reporter gene insilkworm tissues driven by treated promoters of BmCatD. The results of the study are asfollows.The transcription start site of BmCatD was determined by5’-Full RACE-PCR analysis.The site is located at-90bp upstream from ATG. We selected before the transcription startsite about1500bp sequence of the promoter as the research object. There are threeecdysone response elements (EcREs) in BmCatD promoter by Bioinformatics analysis.EcRE1, TTCGATTGACA (-109bp～-99bp); EcRE2, TTTAACTGAAA (-836bp～-826bp) and EcRE3, TTGGAATGAAA (-856bp～-846bp). We inserted the base pointmutations EcREs, which treated by overlapping deletion, into a recombinant AcMNPVvector and performed luciferase assay with a dual-luciferase quantitative assay system.Dual-luciferase activity in fat body suggested that EcREs do participate in the regulation ofBmCatD promoter activity, and the EcRE1near initiation transcription site has themaximum influence.5’ end truncation of BmCatD promoter was performed to study the transcriptionregulation of BmCatD gene affected by promoter length.6promoter sequence fragments ofdifferent lengths (-1363/+91,-1214/+91,-925/+91,-801/+91,-501/+91,-234/+91and-95/+91) were constructed with PCR. We inserted the truncated fragments into therecombinant AcMNPV vector and performed luciferase assay with the dual-luciferasequantitative assay system. These results suggest that the-1214to-925bp region upstreamfrom the transcription start site of the BmCatD promoter confers the highest repression ofBmCatD expression in the fat body. While, there are still some other regulatory elements buthave little effection in the transcription of BmCatD gene in the range of-925to-501, Thenwe deleted the related region,-1214to-925, partially. The partial deletion sequence about-1214to-925of BmCatD promoter was performed with overlap extension PCR. Dual-luciferase activity suggest that the-1071to-1038bp region upstream from thetranscription start site of the BmCatD promoter confers the highest repression of BmCatDexpression in the fat body.