Studies On The Ovo And Otu Genes In Silkworm, Bombyx Mori

The normal development of ovary in Drosophila requires the involvement of a series of genes, among which are the ovo and otu genes that have a close relationship with the ovary development. The silkworm is the typespecies of Lepidoptera and the research reports of genes and molecular mechanism relative to silkworm ovary development are limited. This investigation obtained ovo(Bmovo) of silkworm by silicon cloning on basis of amino acid sequence of Drosophila OVO-B(GenBanka accession:NP₇26971) isoform proteins available to public in GenBank. Three alternatively spliced isoforms of Bmovo gene are acquired in silkworm testis and ovary, designated as Bmovo-1, Bmovo-2 and Bmovo-3, respectively.Homologous blast reflected that the BmOVO-1 and BmOVO-2 contain a Chitinase active site, three clusters of C2H2 type zink finger motif, and BmOVO-3 contains exclusively three clusters of C2H2 type zink finger motif, indicating that possibly BmOVO belongs to transcription factor and involves in energy metabolism. The results of BmN cell localization revealed that BmOVO mainly localized in nucleus. And Real-Time PCR consequences displayed that the aboundence of Bmovo mRNA was highest in silkworm ovary and least in haemoacyte. The results of Western Blotting demonstrated that Bmovo has the capability to express in ovary as well as fat body. This study employed RNAi to downregulate and gene overexpression to upregulate the Bmovo gene expression to investigate the function of Bmovo gene primarily and the outcomes showed that after being injected by double strand RNA(dsRNA), the fertility rate of the experimental group has no conspicuous discrepancy and the hatching rate decreased slightly, however, the egg number produced by the female silkworm decreased obviously, compared with the blank group. The egg number produced by the female silkworm decreased by nearly 15%. Furthermore, the date displayed that the decrease of the egg number produced by the female silkworm and the hatching rate in response to the injection of dsRNA to the female silkworm. The weight of the female pupae and cocoon shell increased by nearly 10% separately, after being injected by dsRNA. The weight of the male pupae increased by nearly 1.0%, however, the cocoon shell increased by nearly 10.8% after being injected by dsRNA. Silkworm overexpressing BmOVO-1 has no effect on the gonad development, the egg number produced by the female silkworm, fertility fate as well as the hatching rate and the G0 as well as the G1 generation are competent to develop normally with natural gonads.The sequence of silkworm otu(Bmotu) gene was achieved by silicon cloning on basis of amino acid sequence of Drosophila Otu-B(GenBanka accession:NP₅11089) isoform proteins. And the results of RT-PCR showed that four alternatively spliced isoforms of Bmotu gene exsisted in silkworm testis, however, only one in ovary. Microarray Database analysis showed that the alternatively spliced isoforms of the Bmotu gene displayed expression distinction among differing tissues. Bioinformatics analysis discovered that proteins encoded by Bmotu are similar to drosophila Otu in structure containing a cysteine proteinase domain (Otu domain), a Tudor domain and a proline-rich motif. The research consequences laid a foundation to elucidate the mechanism of silkworm reproductive development and provide the genetic operation of gonad development with target genes.