荷包红鲤自噬相关基因LC3B的克隆及在重金属镉胁迫下的表达

Molecular cloning,characterization and expression analysis of LC3B from purse red common carp(Cyprinus carpio) exposed to cadmium

微管相关蛋白1轻链3(microtubule-associated protein 1light chain 3,LC3)是哺乳动物细胞中酵母自噬相关基因8(ATG8)的同源物,其LC3B亚型的表达强度与自噬泡数量的多少成正相关,为自噬发生的标志蛋白之一。应用RACE-PCR技术克隆了婺源荷包红鲤LC3B基因的cDNA全长序列,用RT-PCR方法检测了该基因在荷包红鲤主要组织中的表达,并用Real-time quantitative PCR方法检测了该基因在镉胁迫下转录水平的变化,以初步揭示该基因在镉胁迫下的作用。结果表明,荷包红鲤LC3B基因cDNA全长为2 138bp,其中开放阅读框长378bp,编码125个氨基酸,预测分子量为14.73kDa,等电点为8.76。序列比对显示,其编码的氨基酸序列和斑马鱼LC3B具有最高的一致性和相似性,并在系统发育树上聚为一支。该基因在荷包红鲤肌肉、脑、鳃、脾脏、肝脏、血液、头肾、肾脏和心脏组织中均有表达;在镉胁迫下,镉诱导显著影响了该基因在肾脏组织中的转录水平。LC3B基因参与的自噬反应可能参与了抗镉毒性作用。

LC3（microtubule-associated protein 1 light chain 3）,located in autophagosome membranes after processing,serves as a major autophagy marker.In this study,we isolated its LC3B isform from common carp （cyprinus carpio）,one of the most important industrial aquatic animals in China using rapid amplifica-tion of cDNA ends （RACE）.The cDNA of LC3B gene is 2138-nt in length,containing a 378-nt open read-ing frame.The putative LC3B encodes a peptide of 125 amino acids,with a calculated molecular weight of 14.73 kDa and an isoelectric point of 8.76.Multiple sequence alignment showed that the deduced amino acid sequence of common carp LC3B had the highest sequence identity （95.2%）and similarity （96.8%）to that of zebrafish.In a phylogenetic tree based on amino acid sequences from fish,mammalian,avian and am-phibian LC3 genes,the LC3 proteins from different species were divided into three subgroups （LC3A, LC3B and LC3C）.The common carp LC3B formed a phylogenetic cluster with zebrafish,then other verte-brates.The mRNA transcript level of LC3B was detected in muscle,brain,gills,spleen,liver,blood,head kidney,kidney and heart tissues by RT-PCR analysis.LC3B mRNA transcript level in kidney was found significantly elevated after exposure to cadmium.This was markedly affected by exposure concentration, not by exposure time.