Role of novel RNA helicase-like protein in early embryonic development in silkworm, Bombyxmori.

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In order to understand the molecular mechanism of diapause or non-diapause determination in early embryogenesis of the silkworm, mRNA from diapause and non-diapause eggs was compared using the differential display technique. We have cloned the full length of a cDNA by RACE method using a cDNA fragment which was one of the specific cDNA in the non-diapause eggs. The deduced amino acid sequence of the cDNA (RHL) showed homologues of DEAD-box RNA helicase.To clarify the physiological roles of RHL during the early development, we tried to compare between the diapause and the non-diapause eggs until 60 h after the oviposition by RT-PCR. The amplified RHL cDNA was observed through the all stages in the non-diapause eggs. On the other hand, in the diapause eggs, the cDNA was found in 0 to 12 h eggs and vanished in 24 to 60 h eggs after oviposition. Furthermore, if diapause eggs were activated by HCl treatment 24 h after oviposition, the cDNA was observed until 60 h. These data suggest that RHL has an important role in the early embryo of the silkworm. Therefore, we also tried to express of RHL using a coding region of RHL cDNA by pET system. The molecular mass of the recombinant RHL (rRHL) was estimated to be about 68k Da which was the same as the molecular weight predicted from the coding region of RHL cDNA. An anti-rRHL anti sera was prepared by immunization of New Zealand White rabbit using the rRHL as antigen. We found a specific protein in the both the diapause and non-diapause eggs by immunoblot analyses using the antisera. The change of the positive signals of the specific protein was same as the patterns of RHL mRNA expressions. These results are now being undertaken.