Subcellular Localization And Analysis Of Tissue Expression Of Bombyx Mori Serpin-2 And IDGF

Silkworm (Bombyx mori) is an important economic insect and is regarded as a model insect of Lepidoptera. Silkworm genome was sequenced by the whole-genome shotgun method, and released to GenBank in the October of 2003. This is a significant development of silkworm research, and lay the foundation for the study of functional genes of silkworm.Using the method comprised experiment and bioinformatics approaches, we cloned two novel genes of B. mori, and made some basical analysis of these genes in the research. The research process and the main conclusions are present as follows:(1) Serpins are a broadly distributed family of protease inhibitors. In this study, the gene encoding B. mori serpin-2 {Bmserpin-2) was cloned and expressed in E. coli. The Bmserpin-2 cDNA contains a 1125 bp open reading frame (ORF). The deduced protein has 374 amino acid residues, contains a conserved SERPIN domain and shares extensive homology with other invertebrate serpins. RT-PCR analysis showed that Bmserpin-2 was expressed in all developmental stages of B. mori larvae and various larval tissues. Subcellular localization analysis indicated that Bmserpin-2 protein was located in the cytoplasm. Interestingly, real-time quantitative PCR revealed that the expression of Bmserpin-2 in the midgut of susceptible B. mori strain 306 significantly increased at 72 hours post inoculation (hpi) when infected with BmNPV. However, there was no significant increase of the Bmserpin-2 expression in resistant strain NB infected with BmNPV. Thus, our data indicates that Bmserpin-2 may be involved in B. mori antiviral response.(2) In this study, we cloned a novel IDGF gene in B. mori and designated it as BmIDGF. We found that the BmIDGF gene contains eight exons and seven introns, encoding a peptide of 434 amino-acid residues. The protein was predicted to contain one conserved motif of the glycosyl hydrolases family 18 and fall into groupâ…¤chitinases. Sequence alignment showed that BmIDGF shares extensive homology with other invertebrate IDGF. RT-PCR analysis showed that BmIDGF is expressed in all developmental stages of silkworm larvae and various larvae tissues, which was further confirmed by Western blot analysis. Subcellular localization analysis indicated that BmIDGF is located in the extracellular space. We also successfully expressed it in E. coli and further characterized it by SDS-PAGE and mass spectrometry. Taken together, our data suggests that BmIDGF is a chitinase- like extracellular protein, and provides an excellent platform for subsequent studies on its enzyme activity and role in B. mori development.