Functional Analysis Of The Core Regulatory Elements In Cathepsin B Gene Promoter Of Bombyx Mori

Cathepsin B is an important member of cysteine protease, which is a proteolyticenzyme in lysosome. Cathepsin B widely exists in animals, plants, microorganismsand the body. Silkworm cathepsin B (BmCatB) mainly synthesis in the fat body andinvolves in cell apoptosis during the period of larvae and the transformation to thepupa. It plays a very important role in regulating physiological and biochemicalprocessis. Compared with the understanding for the functional mechanism of theBmCatB, little is known about the regulatory mechanism of the BmCatB genetranscription and expression. In this experiment, we analysis BmCatB gene promoterfunction sequence, which provides basis for further research of metamorphosisprocessis in silkworm.1. Sequence analysis of BmCatB gene promoter. First of all, we extract the totalRNA of silkworm. We used5′-Full RACE-PCR to analyse the transcription start siteof BmCatB. DNA sequencing of a RACE-PCR product identified that the accuratetranscription start site is74bp upstream from the translation start site(ATG). Weanalyzed the upstream region of BmCatB using the Gene Quest Module ofDNASTAR. Three putative EcREs: EcRE1(-1035bp~-1025bp), EcRE2(-812bp~-802bp) and EcRE3(-556bp~-546bp); one TATA box(-524bp~-516bp).2. To analysis the transcription regulation of BmCatB gene affected by EcREs.EcRE mutagenesis was performed with overlap extension PCR. Through site-directedmutagenesis of conservative base, we got three single element mutant promoters:EM-1, EM-2, EM-3; three double elements mutant promoters: EM-12, EM-13,EM-23and three elements mutant promoter: EM-123. We inserted the9truncatedfragments into the recombinant AcMNPV vector and detected the dual-luciferaseactivity in fat body with quantitative assay system. The result showed that: All ofthese EcREs are responsible for suppressing expression of BmCatB in the fat body.EcREs play an very important role in the regulation of BmCatB promoter activity, andthe EcRE-3near initiation transcription site has the maximum influence.3. To analysis the transcription regulation of BmCatB gene affected by promoterlength. Different lengths of promoters were generated by a5’ end truncation PCRmethods. For the gene sequence-1424bp~+33bp, gradually shorten the length of thepromoter, we got nine truncation of BmCatB promoters: BF-1(-1,424bp~+33bp),BF-2(-1,283bp~+33bp), BF-3(-1,165bp~+33bp), BF-4(-1,023bp~+33bp), BF-5(-903bp~+33bp), BF-6(-761bp~+33bp), BF-7(-628bp~+33bp), BF-8(-493bp~+33bp), BF-9(-359bp~+33bp). We inserted the9truncated fragments into therecombinant AcMNPV vector and detected the dual-luciferase activity in fat bodywith quantitative assay system. The result showed that: With the continuouslyshortening of the length of the promoter, the promoter activity ratio is on the decline.From BF-4construct the relative luciferase activity decreased obviously. elementscritical for the transcription of BmCatB reside in the142bp region between-1,165bp~-1023bp.