Silkworm Sir2 Gene Expression And Function Of A Preliminary Study

Sir2 (silence information regulator 2) family is a type of conserved depended NAD+ histone/non-histone class III deacetylase which could be found from archaeas to mammals. As a Sir2 protein family, sirtuins play an important role in physiological regulation, such as nervous system, longevity, and so on.A new gene was obtained from pupae cDNA library of silkworm constructed by our laboratory. After sequence alignments analysed in NCBI, it was presumed that this gene coded Sir2-related protein and was named as BmSir2, of which the GenBank accession number was NM001043472. The gene has an ORF of 1164 bp coding a polypeptide of 387 amino acid residues. It contains a SIRT1 domain of SIR2 superfamily with a predicted molecular mass of 43.37 kD and a theoretical pI of 4.94. BmSir2 was cloned into pET-28a(+) vector and transformed into E.coli Rosetta competent cells for expression. The fusion protein was expressed successfully in Rosetta by adding IPTG as inductor. The molecular weight of this fusion protein with His-tag is 47.87 kD. After Ni2+ affinity chromatography, the His-BmSir2 was highly purified and used to immunize a male New Zealand rabbit to obtain the polyclonal antibody against BmSir2 whose titer was larger than 1:12800 by ELISA assay analysis. Total proteins of various developmental stages of silkworm and the different tissues proteins of the fifth instar larvae were extracted for Western blot analysis. The results showed that the distributing of BmSir2 in four stages of silkworm is different with the highest in the egg and the lowest in the fifth instar larvae. In the fifth instar larvae, BmSir2 protein was only found in four different larva's tissues:Silk gland, fat, trachea and head. Subcellular localization of BmSir2 was shown to be mainly presented in cytoplasm of the BmN cells, while less in nucleus.