Studies On The Reproductive Development-Related Gene Bmvlg And Bmvis Silkworm, Bombyx Mori

Primordial germ cells (PGCs) are stem cells originating from primitive germ crest of embyo with the ability to generate multiplex differentiated cells. Which only exists in the embyo for a short time is the precursor of diploid organisms germ cells, and then finally can differentiate into germ cells. The further study of PGCs had a special significance for the study of embyo development in vitro, genes, genomics, drug screening. vasa was first found in Drosophila melanogaster and played an important role in the formation of polar granule and the development of germ cells. It showed that mutunts of vasa can not generate germ cells. vasa was highly conserved in many species and can only exist in germ cells, so it can serve as a molarcule mark for the research of genesis of gamete, the origin,migration ,differentiation of PGCs.The function of vasa-like gene (vlg) was studied for better understanding of the mechanism of the development of silkworm, a fusion expression transgenic vector of Bmvlg and DsRed drived by the promoter of Bmvlg was constructed. Red fluorescence can be seen in transgenic silkworm which showed that we got the transgenic silkworms successfully. Meanwhile, red fluorescence can also been found in unfertilized eggs of silkworm, demonstrating it was a maternal gene. Bmvlg was cloned into pET-28a(+) and expressed successfully. Polyclonal antibodies were raised by immunizing mouse with recombinant proteins purificated by Ni-ions column. Western blotting showed that two strips of 70 kD were seen in both testes and ovary; subcellular localization showed that BmVLG mainly exsited in cytoplasm of BmN cells. The result of RT-PCR revealed that Bmvlg expressed with no significance difference in the gonad of both 4th and 5th in silkworm. All of this studies laied a foundation for further research on Bmvlg in silkworm.Vis (Vismay) belongs to TGIF superfamily, mutants in Drosophila melanogaster revealed that they can survive but males were all sterility, so it can regard as meiosis arrest gene. A 847 bp fragment of Bombyx mori vis (Bmvis) was cloned by RT-PCR on the base of silico cloning as well as structure analysis and phylogenetic analysis. Expression patterns analysis showed that Bmvis can only be detected in testes. Subcellular localization showed that BmVis was located both in cytoplasm and nucleus, but mainly in nucleus. The study lays a foundation for further research on the function of BmVis in silkworm.