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Molecular characterization of heat shock protein 70 promoter from the silkworm, Bombyx mori
- 언어ENG
- URLhttps://db.koreascholar.com/Article/Detail/290762
Recently Transgenesis was achieved in Bombix mori. For stable and effective transgenesis in B.mori, B.mori cytoplasmic actin gene (BmA3) promoter was used to expression of marker gene, the green fluorescent protein(GFP). Green fluorescent protein expression for selection of transformants was visible in all larval, pupal, and adult tissues but, unexpectdly, was not detectable in embryos. So, it spend times and money on rearing of silkworm. Furthermore, the BmA3 promoter is predominantly active in the midgut, which makes it difficult to reliably identify transformants since autofluorescence of many insect foods can mask low-level fluorescence and only allows the detection of strongly expressing individuals with potentially multiple insertions. Therefore, we need more intensely promoter than BmA3 promoter for selected by expression of GFP in embryos and selected by reliable expression of GFP in larvae. We performed dot blot hybridization to develop strong promoter. Nine differentially expressed clones were isolated and we focused one clone of them which has high similarity with heat shock protein 70 gene from D.melanogaster. We named it as bHSP70 (Bombyx mori heat shock protein 70). Expression from the hsp70 promoter was strong and heat shock-dependent. And Drosophila hsp70 promoter appears useful for regulating expression of Exogenous DNA. So, we analyzed transcriptional activity of promoter with bHSP70 gene by using dual luciferase assay system. bHSP70 promoter has about 264 folds more intensely than BmA3 promoter. Also, when bHSP70 promoter treated heat shock(42℃), transcriptional activity incresed 2 times more than normal condition. Therefore, we suggest that bHSP70 promoter is more effective candidate for stable transformation and selection of transformants.
