The Study On Cloning, Sequence Analysis And Gene Expression Of Mago Nashi Gene In The Silkworm, Bombyx Mori

Germ plasm in many species formed during oogenesis ,it is a special cytoplasmic component. cells without the germ plasm can't develop into germ cells. Mago nashi ,a biological reproductive gene in Drosophila, plays an important role in assembling and formation of germ plasm. The protein encoded by Mago nashi gene can recognize and bind some RNA which decide the fate of germ cells .This RNP complex can guide germ plasm assembled correctly. From the lower eukaryotic like yeast to the higher mammals such as homo-sapiens,in which Mago nashi gene has the homology which has very high similarity with it. Mago nashi gene plays a conservative and important role in biological evolution.To further clarify the Silkworm reproductive and developmental mechanisms, based on the analysis of the silkworm genome ,the large scale EST datas and microarray data ,this paper analyzed the homology and Spatio-temporal expression of the key gene which had been reported to determine the formation of germ plasm; The cloning and expression of Mago nashi gene have been done with a view to understand the function of the gene, the results obtained are as follows :1 The homology of reproductive genes playing essential roles in Drosophila germ plasm formation in the silkworm:Downloaded 18 reproductive genes playing essential roles in germ plasm formation in the Drosophila from FlyBase (aub, spir,nos, tor, tud,vas,pum,par-1,capu,stau,osk,valois,germ-cell less,polar- granule component,mtlrRNA, mago nashi, tsunagi,exuperantia). Used BLAST to compare them with silkworm genome data, excepting oskar, valois,germ-cell less,polar granule component, mtlrRNA, the remaining 13 genes in Silkworm have homologous gene. According to the gene array and the gene expression microarray data to analyz the expression patterns of the predicted genes which may have influence on the formation of germ cells. Five of these genes have significant differences in expression of paroecious development in later stage, the expression in femina is significantly stronger than maleness; in the paroecious development process four of these genes have no difference in expression; Three of the genes differentially expressed only in gonad; six genes expressed not only in gonad,but also in other tissues and organs.2 The cloning and expression pattern of Bmmago gene in Bombyx moriFor the expression analysis of Bmmago gene, we cloned the coding sequence of the Bmmago gene by RT-PCR,then the clone sequenced. The Bmmago gene, a single copy locating on the genome, spans 1611bp and is comprised of 2 exons and 1 intron. The gene comprised an open reading frame of 441bp,and the encoded protein was 146 amino acids. The expression of Bmmago gene was found in all kinds of tissues,organs and all stages,thus the gene is a non-specific expression gene.3 The expression of Bmmago gene in E.coliIn order to further research of the characteristics and function of the encoding protein, a prokaryote express vector, pET-Bmmago, containing the complete ORF of Bmmago, had been constructed. The recombinant plasmid was induced by IPTG ,then performed the SDS-PAGE, we found the recombinant protein expressed. Comparing with the reference, between the marker 31KD-20.1KD, recombinant protein shown a clearly new band. This molecular weight correspond the predicted protein size. Further analysis revealed that the recombinant protein in the form of inclusion bodies.