In Situ Hybridization On Vasa Gene Of Silkworm Bombyx Mori.

Frozen sections to silkworm(Bombyx mori) eggs of different developmental stages have been procured. In addition, a lot of pictures have been obtained by a series of frozen sections of different stages. These pictures show respective characteristic of these stages. On the one hand, the results conform and complement the ancestral theories on this study, on the other hand, it is believed that the studies provide a valuable basis on further molecular biology research of silkworm(Bombyx mori), e.g. we have to be acquainted with the normal morphology before the study on gene function.Some continuous frozen sections of 35 h and 48 h embryos have been procured. The results tell us that the embryos keep the cubic shape till the formation of the whole cuticle. Later, the shape are almost keeping cylinder till hatch. Many factors such as stages, methods, personal developmental difference and the protocol could impact the analysis of the results. Therefore, it is better to consult as many as possible references when we research into the embryonic development of silkworms.Vasa is one of the fateful elements of germ line, which is conserved widely in many species. Since it has been first reported in Drosophila, vasa homologues have been identified in many other insects, nemotode (C.elegans), frog(Xenopus laevis), primary eukaryotic organism, plants and mammalian in succession. In our research, a silkworm vasa cDNA has been cloned, which is used as the probe to hybridize in silkworm embryos and gonods after labelled with DIG It shows that silkworm vasa expresses at the sites where the germ line cell exists and the future gonods will occur. On the basis, silkworm vasa gene has been studied by in situ hybridization(ISH) on gonads. The ISH results show that silkworm vasa expresses only in germ cell lines (e.g. oocyte, spermatocyte), implying the important role silkworm vasa plays in the early development of embryos and the differetiation of germ line.In conclusion, silkworm vasa plays an important role in the determination of the germ line.What's more, the optimization of ISH has been tested.The findings show that many factors impact on the experiment results such as materials, fixation, digestion of enzymes, temperature of hybridization and washing, as well as dye methods.To sum up,the establishment of the ISH protocol is necessary and important.