Expression And Localization Analysis Of Bmwibg Gene From Silkworm (Bombyx Mori)

A cDNA sequence was screened from a Bombyx mori cDNA library constructed by our labortary, which contained a small Mago-bind superfamily identified after Blast based on NCBI database. This gene was nominated as Bombyx mori Wibg (BmWibg). The BmWibg gene contained one intron and the length of the cDNA is 853 bp, which contained an ORF of 582 bp, encoding 193 amino acid residues with the predicted molecular weight of 21.86 kD and isoelectric point of 9.538.Two primers were designed to amplify the coding region of BmWibg gene from pHelix vector harboring BmWibg gene. The ORF of this gene was cloned into pET-28a(+) vector with EcoRâ… and Xhoâ… digestion. The recombinant plasmid was transformed into E.coli BL21(DE3). PCR and digestion with EcoRâ… / Xhoâ… showed that the designed fragment was inserted correctly. The recombinant plasmid was sequenced and the result indicated that a recombinant expression plasmid was constructed successfully. Recombinant protein was expressed successfully in E.coli BL21(DE3) after induced by IPTG, the weight of the protein was consistent with the theory value by MS analysis. Most of recombinant protein was soluble and was purified with metal-chelating affinity chromatography. Polyclonal antibody were generated by immuning a New Zealand rabbit with recombinant protein and the titer of it was over 1:25600, measuring by ELISA. The total protein and RNA were extracted from silkworm four different stages and the fifth instar larva eight tissues. The expression level of BmWibg was analyzed by Western blotting and ELISA and the transcription level of BmWibg was analyzed by RT-PCR. The results of RT-PCR showed that the BmWibg gene transcript was highest in spermary of fifth instar larva, and highest in egg and lowest in moth. The results of Western blotting showed that the BmWibg existed in silk gland, spermary and head, and also existed in larval, pupa and egg. The results of ELISA showed that BmWibg protein was highest in egg and lowest in moth, and highest in the silk gland and spermary of fifth instar larva. The three methods analyzed the distribution of BmWibg separately from transcription level and expression level, the results were basically consistent. Immunocytochemistry in BmN cells showed that the protein just existed in cytoplasm. We suggested that BmWibg gene possibly was critical gene in the generation differentiation of early Bombyx mori. These results laid a good foundation for further studies on BmWibg gene.