Molecular Cloning, Expression And Localization Analysis Of A Hypothetial Gene Bm-P312 From Silkworm (Bombyx Mori)

We screened a novel gene with an open reading frame (ORF) of 870 bp encoding 289 amino acids residues from the silkworm pupae cDNA library constructed by our laboratory. The GenBank accession number is DQ237560.After blasting the sequence homology in the NCBI datadase, we identified it was an unknown gen(ehypothetical gene) in the Bombyx mori and nominated the gene as Bm-P312(Bombyx mori-P312).We also take genome alignment between Bm-P312 cDNA and silkworm genome, and the results showed that this gene includes only one exon.After the Blast based on NCBI database, we found the predicted amino acid sequences of Bm-P312 share no similarity to known protein and only with low homologous to certain hypothetical protein or unknown protein.According to the ORF of Bm-P312, two primers were designed to obtain the coding region of Bm-P312 gene. Bm-P312 gene was ampified and cloned into pET28-a(+) vector digested with EcoRâ… and Xhoâ… . The recombinant plasmid was transformed into E.coli BL21(DE3). PCR and digestion with EcoRâ… /Xhoâ… showed that the designed fragment was inserted correctly. Recombinant protein was expressed successfully in E.coli BL21(DE3) induced by IPTG with the final concentration of 1mM. The analysis of SDS-PAGE showed that the fusion protein His-Bm-P312 was expressed in BL21 with 37 kD (Including 3.8 kD His tag and 26.9 kD Bm-P312 protein) that was accorded with the theory value and the most recombinant protein was mainly soluble .The recombinant protein was purified by metal-chelating affinity chromatography. Polyclonal antibody was generated by immuning New Zealand rabbit with recombinant protein and the titer was larger than 1:12800, measuring by ELISA. Western blooting validated the antibody was specific.we located the Bm-P312 by Immunocytochemistry in Bm5 cells and the results indicated that the protein mainly existed in the cytoplasm.We analyzed the transcription level of Bm-P312 mRNA in different stages of silkworm and different tissues of the fifth instar larva. These results suggested that Bm-P312 had a high transcription level in silkworm's pupa,genital glands and brain. The total proteins was extracted from different stages of silkworm and some tissues of the fifth instar larvae for immunoblotting. The results showed that Bm-P312 protein highly expressed in pupa,genital glands,brain and silk gland. These two results had an obvious consistency.According to the experiment, We proved that Bm-P312 gene has the highest transcriptional and expression level in the genital glands of Bombyx mori. Considering the result of its subcelluar localization in Epidermal cell of ovary of Bombyx mori ,We presumed that Bm-P312 protein may play some roles in the reproductive development process of Bombyx mori. These results laid a good foundation for the further studies on Bm-P312 structure and function.