罗氏沼虾MrLc3a基因cDNA的克隆及在副溶血弧菌胁迫下的表达分析

Molecular Cloning,Characterization and Expression Analysis of MrLc3a cDNA from Macrobrachium rosenbergii Exposed to Vibrio parahaemolyticus

虾类和果蝇同属节肢动物。果蝇的相关研究表明自噬与免疫关系密切,而虾类自噬机制研究鲜少。微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,Lc3)与自噬基因Atg8同源,其与自噬体的形成密切相关,是自噬活性的标志分子。本研究利用RACE技术克隆了罗氏沼虾的MrLc3a基因的全长cDNA,用RT-qPCR检测了该基因在罗氏沼虾主要组织中的表达量;并研究了正常和副溶血弧菌感染两种情况下MrLc3a基因和免疫基因Relish的表达变化情况,为其在病害防御方面的应用提供了前期数据。试验结果表明:MrLc3a基因全长653 bp,其中包括195 bp的5'-UTR、378 bp的ORF开放阅读框和80 bp的3'-UTR,共编码126个氨基酸;序列比对结果显示,其编码的氨基酸序列和南美白对虾Lc3a编码的氨基酸序列具有较高的同源性,并在系统发育树上聚为一支;RT-qPCR结果显示,MrLc3a基因在罗氏沼虾各个组织均有表达,其中在脑、鳃、胃中的表达量较高,在肝胰腺和性腺中的表达量较少;副溶血弧菌感染罗氏沼虾后显著影响了MrLc3a和Relish基因在罗氏沼虾肝胰腺组织中的转录情况,MrLc3a和Relish基因随时间变化都呈现出先上升后下降的趋势,表明MrLc3a基因通过参与细胞自噬过程而参与了免疫反应。

Macrobrachium rosenbergii and Drosophila melanogaster all belong to the phylum arthropoda.Previous works in Drosophila melanogaster had revealed that autophagy is closely related to immunity,whereas there are few reports on the autophagy of shrimp.As a molecular marker of autophagy,microtubule-associated protein 1 light chain 3(Lc3),a homologue of the autophagy gene Atg8,is localized in autophagosome membranes after processing.In this study,the cDNA sequence of MrLc3a of Macrobrachium rosenbergii was cloned by RACE-PCR and the expression of MrLc3a in major tissues was detected by RT-qPCR.Also the changes in the expression of MrLc3a and immune gene Relish were studied under the normal and Vibrio parahaemolyticus infected condition.The results showed that the full-length cDNA of MrLc3a gene consists of 653 base pairs(bp),including 195 bp 5'noncoding region(UTR),80 bp 3'non-coding region(UTR)and 378 bp open reading frame(ORF).It encodes a protein of 126 amino acids.The amino acid sequence alignment showed that the Lc3a of Macrobrachium rosenbergii shared high sequence identity with Lc3a of Penaeus vannamei and its phylogenetic analysis was consistent with the traditional morphological classification.The RT-qPCR results showed that the MrLc3a gene was expressed in all tissues,with a higher expression in brain,gills,and stomach,and a lower expression in hepatopancreas and gonads.MrLc3a mRNA and Relish mRNA transcript level in hepatopancreas changed significantly after exposure to Vibrio parahaemolyticus,showing a tendency of rising first then decreasing,which suggested that MrLc3a was involved in the immune response through autophagy.