Screening And Identification Of Hemocyte-specific Promoters In Silkworm, Bombyx Mori

Silkworm (Bombyx mori), which has an important economic value, plays a significant role as a kind of lepidoptera model insect in theoretical research and production application. With the completion of the draft and fine genome sequences and the genome-wide microarray of silkworm, abundant data resource can be utilized for the gene identification and function study of silkworm. The hemocytes of silkworm are of great importance in the metabolism,congenital immunity, metamorphosis and some other areas. Although the related genes of silkworm hemocytes have been preliminary cloning and identification, some issues about the research and application of these genes remain to be resolved. Herein, the hemocyte-specific promoter used as a tool can promote the hemocyte study based on the improvement of silkworm transgenic key theory and the construction of technology system. Moreover, the hemocyte-specific promoter can be used to discover the related gene function and regulatory network of hemocyte, which provides important theoretical basis for the study of the silkworm hemocytes.The primary goal is to clone and identify hemocyte-specific promoters in B. mori. Three hemocyte-specific expression genes were obtained through tissue microarray data of gene expression and RT-PCR. Then the promoters of hemocyte-specific expression candidate genes were cloned based on silkworm genome database. The recombinant Autographa california multiple nuclear polyhedrosis virus (rAcMNPV) were used as gene transfer vectors. The vectors and flow cytometry method (FCM) were applied to analyze the activities and specificities of the promoters in silkworm. The main results are as follows:1.Identification of hemocyte-specific genes of silkwormBy tissue microarray data and tissue expression profiles analysis, three candidate genes were confirmed, including BmCathepsinO, Bmintegrin β2 and BmO4862, which were specifically expressed in hemocyte. BmCathepsinO, belongs to a member of the cysteine protease family, which is mainly involved in lysosome hydrolysis, Bmintegrin β2 is a member of the integrin family in the silkworm, participating in the cell surface adhesion and immune function. Bm04862 is a noval gene that has not been reported.2.Cloning and activity analysis of the BmCathpsinO promoterThe 5’terminal-truncated BmCathepsinO promoter sequences about 2000 bp has been cloned from Dazao genomic DNA, pBmCatO2k, which was based on silkworm genome database. The recombinant autographa california multiple nuclear polyhedrosis virus (rAcMNPV) were used as gene transfer vectors. It was found that the cloned sequences pBmCatO2k possessed hemocyte-specific activity. Then, the activity analysis of the five different BmCathepsinO promoter sequences, was conducted, including pBmCatO2k, pBmCatO1.5K., pBmCatO1K, pBmCatO0.5K and pBmCatO0.2K.The results showed that the critical site of the promoter was from-80 bp to+76 bp and the upstream transcription start site from-333bp to-80 bp of BmCathepsinO gene contained the tissue specificity regulatory elements, which can control the tissue specificity.3.Cloning and activity analysis of the Bmintegrin p2 promoterThree Bmintegrin β2 promoter sequences with different sizes from the upstream transcription start site of Bmintegrin β2 were firstly cloned, including pBmIntβ22k, pBmIntβ23k, pBmIntβ25k. It was found that all of the three promoters were inactive because they cannot drive the DsRed expression in BmE-SWU3 cell and organs of silkworm. Then, the promoters containing the first intron, i.e. pBmIntβ23kIN and pBmIntβ21.5kIN, were further cloned. As a result, they possessed promoter activity on the hemocyte mediated by virus, while had no activity in the fatbody and silkgland. These results show that the first intron is an active region of Bmintegrin β2 promoter and has an important effect on controlling tissue specificity.4. Bm04862 gene identification and analysis of the promoterThe full-length cDNA sequence of Bm04862, including 819bp, was obtained by the rapid amplification of cDNA ends (RACE), which was predicted to encode a transmembrane protein with 273 amino acid residues. Afterwards, qRT-PCR was applied for its temporal expression profile. It was specifically expressed in hemocytes, and reached a peak at L4M and L5M stage. The cellular localization experiment indicated that it was specifically distributed on cytoplasm and nuclei membrane. The qRT-PCR was also used to monitor the Bm04862 expression after stimulated by E.coli.Bm04862. Its expression dramatically increased after challenged with E.coli for 24h, indicating its potential role in the innate immune system. The 2194bp Bm04862 promoter was cloned, which can drive the red fluorescent protein expression in BmE-SWU3 cell, blood cells, silk gland and fat body. The results of FCM indicated the activity of Bm04862 promoter was 4.8 times higher than that of Pph promoter, which means that pBm04862 is a strong promoter without tissue specificity. It can be speculated that the tissue specificity regulatory elements of Bm04862 may located in the far area of transcription start bit or the characteristics of Bm04862 promoter may be affected by the virus.