Study On Expression And Functional Analysis Of BYB Gene From Bombyx Mori

Y-box proteins are a family of highly conserved nucleic acid binding proteins which exist widely in various organisms from bacteria to human. The high level of conservation of these CSD (cold shock domain) proteins suggests that Y-box proteins play essential roles in biological processes. The full open reading frame (ORF) of Bombyx mori Y-box was obtained from silkworm pupa by RT-PCR and named Bombyx mori Y-box (BYB), of which the accession number was AB098537.1. This ORF contained 780 bp, encoding a polypeptide of 259 amino acids with the predicted molecular weight of 28.67 kD. After cloned into pET-28a(+) and expressed in E.coli BL21 (DE3), BYB was fused with His-tag and purified on chelating columns. The molecular weight of this fusion protein was 32 kD , characterized by SDS-PAGE after being further purified. This result proved that BYB with His-tag was expressed in the right form. The purified fusion protein was used to immunize a male New Zealand rabbit. Then, antiserum was harvested, and IgG was prepared by affinity chromatography using immobilized protein A. BYB in some tissues of the fifth instar larvae, such as head, Malpighian tubule, epidermis, silk glands, seminal glands, gut and fatty body, were extracted and analyzed by Western blotting. The result showed that BYB existed in larva's seminal glands, Epidermis, and so on. And the similar result was obtained when immunohistochemistry analysis was applied to the fifth instar larva and pupa. We also compared the quantity of BYB mRNA in different tissues of the fifth instar larva and different stages of silkworm growth. These results suggested that BYB was selectively expressed in silkworm's seminal glands, epidermis and Malpighian tubule. After subcellular localization with BYB, we found that it mainly existed in cytoplasm under normal conditions. However, after BmN cell had been treated with cold shock of 4℃for 2 h, most BYB proteins distributed in the cytoplasm and the nucleus. This research laid a good foundation of further studies on BYB.