Characterization Of Bmlipase-1Gene Promoter And Analysis Of Its Differential Expression Among Silkworm Strains

Bmlipase-1is a digestive enzyme in silkworm, and its main physiological function isdigestion and hydrolysis of lipids and lipoproteins. However, in the initial stage of BmNPVinfection, Bmlipase-1can inhibit viral amplification, and protect intestinal epithelial cellagainst BmNPV-ODV. Moreover, Bmlipase-1has a tissue-specific expression in midgutand plays a role of resistance to virus in the first immune defense line of silkworm.Thisarticle analyzed Bmlipase-1promoter regulation district, providing basic information forscreening specific promoters that can be used for transgenic silkworm study, but also helpsto further elucidate the transcriptional regulation mechanism of Bmlipase-1;Expressionlevels of Bmlipase-1were determined in various strains of silkworm, to explore therelationship between expression levels of Bmlipase-1and the resistance to BmNPV insilkworm. This article consists of the following two parts:1. Bmlipase-1promoter regulatory sequences are cloned from silkworm genomic DNA,then sequenced and analyzed. According to the prediction of software and using EGFP asreporter gene, we constructed a series of reporter plasmids with different promoter elementsdeletion through Bac-to-Bac baculovirus expression system; transected cells withrecombinant baculovirus, injected silkworm larvae with recombinant baculovirus, anddetermined the expression of EGFP at transcriptional and translational levels, byfluorescence observation, quantitative PCR and ELISA.The cloned sequence covers the first exon (1~52), the first intron (53~883), corepromoter region (-100~-1) and5’-upstream region (-1980~-101), but identifiable TATAbox was not predicted in the area adjacent to the transcription start site. According to theexpression levels of reporter gene, the activity tendency of different length promoterfragments is:-1453~192supreme,-1453~955and-678~192second,-678~955third,-383~955and-81~955fourth,-1980~955and494~955minimum. It is speculated thatthere might be positive regulatory elements in Bmlipase-1promoter’s upstream of-1453~-679,-678~-384and-81~493, in the region of-1454~-1980and494-955there mightbe negative regulatory elements, and in the region of-82~-383there is no significanteffect. Among them,-1453to-679region contains some promoter conserved sequences ofsilkworm midgut tissue-specific related gene, and1CAAT box and a large amount ofGATA sequences, suggested that this region may be associated with Bmlipase-1 tissue-specific expression.-81~493region contains the core promoter, the first exon andthe first intron sequence, and contains a large amount of Pbx-1binding sites, OTCsequences and GATA sequences, speculated that this region has a basic function forBmlipase-1transcriptional regulation.2.6silkworm varieties with different BmNPV resistance levels were chose as materials.To explore the relationship between expression of Bmlipase-1and resistance to BmNPV,Bmlipase-1expression levels of health larval and BmNPV-infected larval were detected inmidgut tissue of different varieties by fluorescence quantitative PCR (qRT-PCR)technology.Bmlipase-1expressions have significant difference among diversified silkwormvarieties, and expression levels are higher in strains provided with stronger viral resistance.In the tested varieties, expression level of Bmlipase-1in NIL·LVR is the highest; p50,CVDAR18, nsd·NIL reduced in turn;892and306are at the lowest level. The averageexpression amount of Bmlipase-1in the5th instar of NIL·LVR was about8.2times morethan that of306. Bmlipase-1gene could be induced after infected with BmNPV, and theinduced expressions are higher in strains with stronger viral resistance than that in strainswith weaker resistance. In the tested varieties, induced expression level of Bmlipase-1inNIL·LVR is the highest; p50second; CVDAR18and nsd·NIL third;892and306are thelowest. The average expression amount of Bmlipase-1in the5th instar of NIL·LVR was12.4times more than that of306, after infected by BmNPV. It is speculated that expressionlevels of Bmlipase-1have certain relevance with the resistance against BmNPV amongsilkworm strains.