Characterization And Analysis Of DRP And SSP Gene Of Bombyx Mori

Silkworm, Bombyx mori, is an important economic insect and the model insect of Lepidoptera. Silk industry has developed for more than 5000 years in China, and made a great contribution to the life and economy of China. Meanwhile, research on functional genomics of silkworm lay the foundation for people to comprehend the biological heredity and molecular regulation of silkworm exactly and it also can help us to elucidate the biological phenomenon and promote the outcome of silk industry.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 this research. The main conclusions are presented as follows:1. Using the in silico cloning method, we cloned the DRP (death-related protein) gene cDNA and analysed with bioinformatics tools, the result shows that the DRP cDNA is 761 bp in full length, which contains a 306bp ORF and includes two exons. The deduced protein has 102 amino acid residues with the predicted molecular weight of 21KD and locates on chromosome 20. The protein shows high degrees of identity with that of some homologous protein from other species. The result was confirmed by RT-PCR and sequenced. This gene has been registered in GenBank under the accession number FJ447481. Through RT-PCR analysis, we found this transcript was present in all tissues and all developmental stages. The DRP gene was cloned into prokaryotic expression vector pET30a to construct a recombinant plasmid pET30a/DRP. DRP was expressed under the induction of IPTG and identified by SDS-PAGE. The molecular mass of expressed GlcAT-S is a about 21kD.2. Using the in silico cloning method, we obtain SSP(salivary secreted protein). The cDNA full sequence of SSP gene was obtained in GenBank, then we cloned and sequenced it. This gene has been registered in GenBank under the accession number FJ447481. Using pET-30a system to express and identify the expressed protein through Western blot analysis, and then we prepared antiserum with the identified protein. Previously we constructed a B. mori strain BC8, which retains anti-BmNPV properties of NB and is near-isogenic to 306 (susceptible to BmNPV). Here, to further investigate the B. mori anti-BmNPV mechanism, we carried out real-time PCR analysis in these strains, and through RT-PCR analysis, we found this transcript was present in all tissues and all developmental stages in NB and 306 strains. The Quantitative real-time PCR result shows that genes encoding SSP protein, which was up-regulated in the midgut of BC8 within Oh to 72h post inoculation (hpi). Interestingly, the transcript levels of SSP gene significantly decreased after 24 hpi, then maintained a relative higher level. However, the change was not obvious in near-isogenic to 306 (susceptible to BmNPV). This may indicate that SSP plays a role in the resistance against BmNPV and may be involved in B. mori immune response against BmNPV infection.