香鱼 ApoC－Ⅰ的克隆、序列分析及其镉胁迫下的表达

摘要

ApoC-Ⅰ( Apolipoprotein C-Ⅰ) is the smallest member of the apolipoprotein family , which plays important roles in lipid uptake, secretion and transport .Here, we obtained the full-length cDNA sequence of ayu ( Plecoglossus altivelis ) ApoC-I gene ( PaApoC-Ⅰ) through random sequencing of cDNA library .The cDNA sequence of PaApoC-Ⅰis 574 nucleotides , consisting of a 267 bp open reading frame that encodes a protein of 88 amino acids with a deduced molecular mass of 10.08 ku.The N-terminus of PaApoC-Ⅰcontains a predicted signal peptide of 20 amino acids .Multiple sequence alignment of complete amino acid sequences showed that PaApoC-Ⅰshared the highest amino acid sequence identity (67.0%) with that of rainbow trout ( Oncorhynchus mykiss) . Phylogenetic tree analysis revealed that ApoC-Ⅰfrom mammals, reptiles, amphibians and fish formed different clusters , and PaApoC-Ⅰfalled into the fish ApoC-Ⅰcluster and was most closely related to that of rainbow trout .Genome structure analysis showed that PaApoC-Ⅰcontained three exons and two introns .Quantitative real-time PCR ( qRT-PCR) analysis showed that PaApoC-ⅠmRNA had the highest expressional level in ayu liver .Upon cadmium (Cd) exposure, PaApoC-I transcripts were significantly up-regulated in ayu liver, spleen, kidney and intestine, and were significantly down-regulated in ayu gill.In summary, our present study demonstrated that a tight relationship between the PaApoC-Ⅰexpression and Cd stress , and it can be a potential biomarker for Cd exposure in aquatic or-ganisms.The results would provide the basic data for further studying the function of fish ApoC-Ⅰand its mechanism in fish response to heavy metal stress .%载脂蛋白C－Ⅰ（ Apolipoprotein C ， ApoC－Ⅰ）是载脂蛋白家族中分子质量最小的成员，在脂类摄入、转运和分泌中发挥重要作用。通过cDNA文库随机测序获得了香鱼（ Plecoglossus altivelis） ApoC－Ⅰ（ PaApoC－Ⅰ）全长cDNA序列。 PaApoC－Ⅰ由574个核苷酸组成，包含一个长度267 bp的开放阅读框，编码88个氨基酸，预测分子量为10．08 ku， N－末端1～20位氨基酸残基为信号肽序列。氨基酸序列多重比对结果表明， PaApoC－Ⅰ与虹鳟（ On－corhynchus mykiss） ApoC－Ⅰ同源性最高，为67．0％；系统进化树分析表明，哺乳动物、爬行动物、两栖类和鱼类的ApoC－Ⅰ分别成簇， PaApoC－Ⅰ位于鱼类ApoC－Ⅰ簇中，且与虹鳟ApoC－Ⅰ进化相关性最高。基因组结构分析表明， PaApoC－Ⅰ基因组DNA序列由2个内含子和3个外显子组成。实时荧光定量PCR （ quantitative real－time PCR， qRT－PCR）结果显示， PaApoC－ⅠmRNA表达量在香鱼肝组织中最高，镉胁迫后在肝、脾、肾和肠中显著上调，在鳃中表达量显著下调。综上， PaApoC－Ⅰ的表达与镉胁迫密切相关，可能作为水环境中重金属镉污染的生物分子标记。研究结果为进一步研究鱼类ApoC－Ⅰ的功能及其在重金属胁迫中的作用机制提供了基础资料。