太平洋牡蛎不同温度处理下转录组分析与类B3GALT1基因的克隆及表达

Transcriptome analysis of different temperature stress in Crassostrea gigas and cloning and expression of B3GALT1-like

中文关键词:  太平洋牡蛎  转录组  诺如病毒  类B3GALT1基因  组织表达

英文关键词:Crassostrea gigas  transcriptome  norovirus  B3GALT1-like gene  tissue expression

中文摘要:

      目的 探究不同温度处理下太平洋牡蛎中诺如病毒结合受体合成通路上响应的基因, 筛选后进行克隆和表达规律的研究。方法 对牡蛎分别进行高温(25 ℃)及低温(5 ℃)处理, 取其鳃和消化腺两种组织, 提取RNA后进行转录组测序与分析。选取诺如病毒结合受体合成通路上规律性响应温度处理的基因作为目标, 对目标基因进行克隆、原核表达与免疫印迹鉴定。通过实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qRT-PCR)分析该基因的组织表达和季节表达规律。结果 类β1,3半乳糖转移酶1 (B3GALT1)基因(GenBank LOC117683256)在鳃组织低温组的多个取样点均出现显著上调。扩增得到该基因1089 bp的编码区(coding sequence, CDS)。在25 ℃、异丙基-D-硫代半乳糖苷(isopropyl beta-D-1- thiogalactopyranoside, IPTG)诱导8 h后, 出现大小约为84.9 kDa的蛋白条带, 该条带与抗MBP标签抗体、抗人B3GALT1抗体均能发生特异性结合。类B3GALT1基因在鳃组织中大量表达, 且低温时的表达量显著高于高温(P<0.01)。结论 太平洋牡蛎类B3GALT1基因的表达量受温度影响, 表达的蛋白与人类β1,3半乳糖基转移酶具有相似的免疫原性; 类B3GALT1基因的组织表达和季节表达规律在一定程度上与诺如病毒爆发的季节性相符合。本研究为深入探索牡蛎季节性富集诺如病毒分子机制提供基础。

英文摘要:

      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.