家蚕未知基因Bm-P312的克隆表达及定位研究

We got a novel gene with an open reading frame(ORF)of 870bp and encoding 289 amino acids 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 unknow gene in the Bombyx mori andnominated the gene as Bm-P312（Bombyx mori-P312）.According to the ORF of Bm-P312, two primers were designed to obtain the coding region of Bm-P312 gene. We ampifyed the Bm-P312 gene and constructed the rocombinant plasmid pET-28a-Bm-p312, The recombinant plasmid was transformed into E.coli BL21(DE3). and the recombinant protein was expressed in the E.coli BL21(DE3) induced by IPTG, then the recombinant protein was purified and immuned the rabbit to get antiserum.we located the Bm-P312 by Immunocytochemistry in Bm5 cells , 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 and seminal glands and brain . The total proteins was extracted from different stages of silkworm and some tissues of the fifth instar larvae, such as brain, epidermis, silk gland, midgut, Malpighian tubule, fat body, stigma and analyzed by Western blotting. The results showed that Bm-P312 highly expressed in pupa、seminal glands、brain and silk gland. The two results has an obvious consistency.These results laid a good foundation of further studies of Bm-P312.According to the experiment, we audaciously presumed that Bm-p312 may play some role in the reproductive development process of Bombyx mori. But the function of Bm-p312 should be demonstrated by further study.