Transcriptome analysis of different temperature stress in Crassostrea gigas and cloning and expression of B3GALT1-like
Objective To explore the genes that respond to the synthesis pathway of norovirus binding receptors in Crassostrea gigas under different temperature stress,screen them,and study their cloning and expression patterns.Methods Crassostrea gigas were treated at high temperature(25℃)and low temperature(5℃),and two tissues of gills and digestive glands were extracted for RNA extracted for transcriptome sequencing and analysis.Selected genes on the synthesis pathway of norovirus binding receptors that responded to temperature treatment as targets,clone,express in prokaryotic cells,and identified by Western blotting.The tissue expression and seasonal expression pattern of the gene were analyzed by quantitative real time-polymerase chain reaction(qRT-PCR).Results The β1,3 galactosyltransferase-like 1(B3GALT1-like)gene(GenBank LOC117683256)was significantly upregulated in multiple sampling sites in the gill group.The 1089 bp coding sequence(CDS)region of the gene was amplified.After 8 h of induction of 25℃,isopropyl beta-D-1-thiogalactopyranoside(IPTG),a protein band of about 84.9 kDa appeared,which was specifically bound to both anti-MBP tag antibody and anti-human B3GALT1 antibody.B3GALT1-like genes was abundantly expressed in gill tissue and was significantly higher at low temperature than at high temperature(P<0.01).Conclusion The expression level of B3GALT1 gene in Crassostrea gigas is affected by temperature,and the expressed protein has similar immunogenicity to human β1,3 galactosyltransferase.The tissue expression and seasonal expression patterns of the B3GALT1 gene are somewhat consistent with the seasonality of norovirus outbreaks.This study provides a foundation for further exploration of the molecular mechanism of seasonal enrichment of norovirus in Crassostrea gigas.
万方数据
维普
