Studies On The Reproductive Development-Related Genes Bmvlg And Bmachi In Silkworm, Bombyx Mori

The occurrence and development of germ cell are the essential affairs for sexual reproductive animals in fetation, and play a very important role in the development of organism .Drosophila vasa gene encodes the very important protein exists in the polar plasma.The mutation of vasa gene can contribute to the embryo being not able to form the germ cell.The VASA protein featuring as the molecular maker, is specific expression in gonad . Drosophila achi and vis gene encode the highly related members of the TGIF subgroup of TALE homeodomain proteins.In the absence of vis and achi, spermatogenesis is blocked prior to the meiotic divisions with no signs of differentiation, placing these genes in the category known as male meiotic arrest genes.Bombyx mori is the representative of Lepidoptera insects.In order to approach the molecular mechanisms of reproductive development in silkworm, the promoter of Bmvlg gene(Bombyx mori vasa-like gene)was cloned and sequenced.An expression vector was constructed,in which the reporter gene DsRed was driven by Bmvlg promoter. The research resulted that Bmvlg promoter could drive the expression of DsRed temporarily in BmN cell as well as in oosperm;Gaining four cDNA fragments attributed to the alternative splicing occured in vlg gene utilizing RT-PCR technique.It is presumed that there might be various species of alternative splicings happened on Bmvlg gene;Getting the Bmachi cDNA fragments by RT-PCR technique,the result showed that Bmachi expressed in both ovary and testis,however,it might exist different splicing modes and abundances among male and female; The fusion protein BmAchi was successfully expressed in BL21.Polyclonal antibodies were generated by immunizing SPF mice with purified recombinant protein.The result of western blotting showed that the polyclonal antibodies could bind His-BmAchi specifically;Designed and synthesized Achi-dsRNA and a preliminary study were taken on the function of Bmachi gene by RNAi.The silk moth injected Achi-siRNA-2 (TCGCGTCGCTAACAGACAA)or Achi-siRNA-1(GATAGTGACAATACGGAAA) when they were 3-4th instar larvae .The experiment showed that the rates of nonfertilized eggs which were layed by silk moth after mating and normal spawning were 23% and 90%,respectively.Therefore,Bmachi belonged to the male meiotic arrest genes, meanwhile, the expression level of Bmachi caused the obvious effects on gametogenesis and differentiation.