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

为了解家蚕早期胚胎发育滞育与非滞育的分子机制，采用差异显示技术对滞育卵与非滞育卵的mRNA进行了比较。利用非滞育卵特异性cDNA片段，通过RACE方法克隆了其全长cDNA。为了阐明RHL在早期发育过程中的生理作用，本研究采用RT-PCR技术对滞育卵和非滞育卵进行了比较，直至产卵后60 h。扩增的RHL cDNA在非滞育卵的各个发育阶段均有表达。在滞育卵中，该cDNA在产卵后0 ~ 12 h的卵中出现，在产卵后24 ~ 60 h的卵中消失。此外，如果滞育卵在产卵后24 h被HCl处理激活，则cDNA直到60 h才被观察到。这些数据表明，RHL在家蚕早期胚胎中具有重要作用。因此，我们还尝试了利用RHL cDNA的编码区通过pET系统表达RHL。重组RHL（rRHL）的分子量约为68 kDa，与RHL cDNA编码区预测的分子量一致。以rRHL为抗原免疫新西兰白色家兔，制备抗rRHL的抗血清。利用抗血清进行免疫印迹分析，在滞育卵和非滞育卵中均发现了一种特异性蛋白。特异性蛋白阳性信号的变化与RHL mRNA表达的变化规律一致。目前正在取得这些成果。

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.