Characterization And Analysis Of V-ATPase-B And 6PGL 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 make a great contribution to the life and economy of China. 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 this research. The research process and the main conclusions are present as follows:(1)Using the in silico cloning method, we cloned the V-ATPase B Subunit, this gene was a well conserved protein in many animals. The RT-PCR result shows that this gene was expresed in almost all tissues of B. mori. Larvae of highly susceptible silkworm strain 306 and resistant silkworm strain NB were fed with mulberry leaf that treated with Bombyx mori nucleopolyhedrovirus (BmNVV). Total RNA was isolated from the midgut at 24, 48 and 72h after BmNYV infection. The Quantitative real-time PCR result shows that at 24h p.i. expression level of V-ATPase-B in the resistant strain was much higher than in the the susceptible strain. This may indicate that V-ATPase play a role in the resistance against UmNPV. Then the expression level of V-ATPase-B decreased rapidly at 48h p.i. and 72h p.i., the expression was at a very low level.Meanwhile, the putative protein was expressed in E. coli and the fusion protein with the molecular weight about 60 kD was obtained. The fusion protein was purified and then used to immunize the New Zealand white rabbit and the antiserum was collected. The immunohistochemistry analysis on midgut of silkworm larvae shows that positive signanls of the V-ATPase protein were detected in the midgut goblet cell apical membrane. Goblet cells have a cavity, which is formed by invagination of the apical membrane, also have positive signanls.(2)Using the in silico cloning method, we cloned the 6PGL gene and analysed with bioinformatics tools, the result shows that the 6PGL cDNA comtains a 702bp ORF and includes four exons and three intorns. The deduced protein has 233 amino acid residues, with the predicted molecular weight of 26KD , isoelectric point of 5. 41, and contains conserved NagB domains. This gene has been registered in GenBank under the accession number EF198104. The result was confirmed by RT-PCR and sequenced. The putative protein was expressed in E. coli and the fusion protein with the molecular weight about 30 kD was obtained.