Molecular cloning and expression analysis of glucose-regulated protein 78 (GRP78) gene in silkworm Bombyx mori

Abstract

GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full-length cDNA of GRP78 from silkworm Bombyx mori (BmGRP78). It is 2645 bp, including an open reading frame (ORF) of 1977 bp encoding a polypeptide of 658 amino acids. We sequenced the ORF sequence from genomic DNA, and found only one intron (104 bp) existed in Bmgrp78 gene structure. The deduced amino acid sequence of Bmgrp78 carries the ER retention signal KDEL at its C-terminus and is highly homologous to GRP78 of Fenneropenaeus chinensis (83.59%) and Homo sapiens (80.27%). RT-PCR analysis shows that Bmgrp78 is ubiquitously expressed in tissues of silkworm. Heat shock over 35°C notably enhanced the expression of Bmgrp78 in head tissues. In response to starvation, the transcript level of Bmgrp78 in head tissues was 2.8-fold (at WS stage) and 3.2-fold (at SD1 stage) higher than the control level, but it remained stable in the midgut tissues. After silkworms were challenged by bacteria, the relative expression level of Bmgrp78 in midgut was up-regulated and reached maximal level at 24 hour after injection, and remained higher level than that of controls at about 48 hour post challenge. We infer that BmGRP78 may play important roles in chaperoning, protein folding and immune function of silkworm.