Expression And Regulation Study On Silkworm (Bmbyx Mori) β-N-Acetylglucosaminidases

During the molting process, insects couldn’t grow and develop normally if thedegradation and formation of cuticule was disturbed. Chitin is an important component ofinsect cuticle. So the biosynthesis and degradation of chitin are closely related with insectgrowth and development. β-N-Acetylglucosaminidase (GlcNAcase) is a key enzyme in thechitin decomposition process. The silkworm(Bombyx mori)is not only a model organismfor lepidopteran insects, but also economically important in silk industry. And they are thematerials of bioreactor and new insect industry. To further investigate the geneticrelationship with other related enzymes involved in the species, espression in varioustissues and promoter anctivity of silkworm β-N-Acetylglucosaminidase enzymes(BmGlcNAcases), the present study was addressed. The main results were shown asfollows:We studied the BmGlcNAcases using bioinformatics methods. The results show thatBmGlcNAcase1，BmCHiGlcNAcase and BmFDL are closed related with GlcNAcases inother insects,but BmGlcNAcase2， BmGlcNAcase3， BmGlcNAcase4have closelyrelationship with hexosaminase in mammalian rather than insects. Sequence alignmentshowed these proteins have highly conserved catalytic domain.Using dual-spike-in qPCR method, we examined the expression of BmGlcNAcases invarious tissues of silkworm as well as expression changes after stimulation with ecdysone.The expression levels of the BmGlcNAcase genes varied in different tissues, and all werehigher48h after exposure to ecdysone. The espression level of all genes were increasedsignificantly (p<0.05) except BmFDL. We conclued that moulting hormone regulate theexpression of BmGlcNAcases. In order to study the mechanisms underlying regulation of BmGlcNAcase1, functionalanalysis of the promoters of BmGlcNAcase1from Bombyx mori was performed in thisstudy. Recombinant Bacmid containing serially truncated promoter fragments ofBmGlcNAcase1were transfected into Sf9cells, than collected AcMNPV Viruses from Sf9cells which were used to inject silkworm. Three days later, activities of luciferases weremeasured in various tissues using the dual-luciferase reporter assay system. The resultsshow that BmGlcNAcase1promoter activity was higher in the blood, skin, fat body andlower in the silk gland and Malpighian tubules. This suggest that BmGlcNAcase1promoteris tissue specific. Although slightly different in various tissues, when the region-547bp to-223bp was deleted, the promoter activity was drastically reduced, suggesting that thisregion may be the core promoter region. The BmGlcNAcase1promoter activity wereincreased exposure to ecdysone.The region-547bp to+1bp of BmGlcNAcase1was divided into five sections andincorporated into pGL3-basic plasmids respectively. Luciferase reporter plasmidscontaining serially truncated promoter fragments of BmGlcNAcase1were transientlytransfected into BmN cells with internal control vector pRL-TK. The result show that whenthe region-347bp to-223bp was deleted the promoter activity was drastically reduced.The result demonstrated that the region-347bp to-223bp contains core promoterelements. Analysis of binding factors in the region using TFSEARCH software revealedmultiple transcriptional binding sites such as TATA-box, Hb, BR-C Z and HSF. One orseveal of these factors play an important role in regulating the expression ofBmGlcNAcase1.