Cloning And Identification Of Midgut-specific Promotor In Silkworm, Bombyx Mori

Bombyx mori, domesticated from wild silkworm, is a lepidoptera insect with great economic value. According to literature and excavation, silk industry has a long history of more than5000years and has brought great and far-reaching influence all around the world. Silkworm diseases not only cause economic losses up to$100millions of per year but also affect the development of the silk industry. Midgut is a primary immune-defense organ of silkworm, which is the first infected organization after oral ingestion. of pathogenic microorganisms. Some midgut-specific genes play important roles in the development and immunity of silkworm. So we need midgut-specific promoter in B. mori to study gene function by transgenic technology.The primary goal is to clone and identify a midgut-specific promoter in B. mori. Midgut-specific expression genes were obtained through tissue microarray data of gene expression and RT-PCR. Then we clone the promoters of midgut-specific expression candidate genes based on silkworm genome database, we also analyze the activities and specificities of the promoters in silkworm using trangenic method. The main results are as follows:1. Identification of midgut specific expressed genes of silkwormBy tissue microarray data analysis, we identified six candidate genes which have high expression levels in midgut of silkworm. Further tissue expression and period expression analysis confirmed that BGIBMGA008060and BGIBMGA014298were midgut-specific genes. BGIBMGA008060, a protease of the Bombyx mori aminopeptidase N (BmAPN) family members, can hydrolyse protein or peptide N-terminal amino acid in the larvae. BGIBMGA014298is one of the juvenile hormone binding protein family members.2. Cloning and activity analysis of the BmAPN promoterThe1598bp sequence which containing BmAPN gene exon1, the core promoter region and5’flanking region of midgut-specific gene BmAPN was cloned from Dazao genomic DNA. A transgenic vector expressing EGFP gene was constructed under the control of BmAPN promoter. Transgenic lines were generated via embryo microinjection. Among the two transgenic system obttained, Inverse PCR analysis showed that BmAPN-EGFP expression box inserted in nscaf2952and nscaf2655of15th chromosome in the silkworm genome respectively. Quantitative PCR and Western blotting results show that the EGFP was specifically expressed in the midgut of transgenic lines, which indicated the BmAPN was a midgut specific promoter of silkworm. However, the promoter activity in larvae was higher than that of egg and pupa, while in newly exuviated larvae was higher than that of molt. These result showed that BmAPN is a midgut-specific promoter, which should be conducive to the basic and applied research of silkworm.3. Expression analysis of EGFP gene driven by P2promoter in transgenic silkworm, B. moriA1080bp sequences named P2was cloned on the upstream of BGIBMGA014298gene, which showed the typical structure of the promoter by the prediction online. We construct a transgenic expression vector pBac [P2-EGFP-SV40,3×P3DsRed] by using P2as a promoter and EGFP as a reporter gene, which was further used to conduct transgenic microinjection for verification of the promoter’s characters in individual silkworm larve. We screened transgenic system by fluorescent.inverse PCR showed that P2-EGFP expression box were inserted into the No.4and No.23chromosome in B. mori in two transgenic systems respectively the. Continuous fluorescence observation found that the positive individuals can send out green fluorescence in the larval body surface since the fourth instar.Fluorescence observed in3rd day of fifth instar of transgenic silkworm after anatomy, we can see clearly that the anterior portion of midgut emit strong green fluorescence. Transcription detection show that the EGFP expressed strongly specific in the transgenic silkworm midgut, the western blotting result also revealed that the P2promoter is a midgut-specific promoter consistent with the observed fluorescence. The EGFP expression levels was higher in larve than that in eggs and pupa, in agreement with the expression pattern of endogenous gene BGIBMGA014298.