团头鲂g型溶菌酶基因全长cDNA的克隆与表达分析

Molecular cloning and expression analysis of g-type lysozyme gene from blout snout bream( Megahbrama amblycephala)

为了解g型溶菌酶基因序列特征及其在团头鲂(Megahbrama amblycephala)氨氮胁迫过程中的作用,应用末端快速扩增(rapid amplication of c DNA ends,RACE)技术克隆了团头鲂的g型溶菌酶基因c DNA序列,得到全长719 bp的c DNA序列,包括71 bp的5'末端非翻译区(untranslated regions,UTR)、90 bp的3'UTR和558 bp的开放阅读框(open reading frame,ORF);氨基酸相似度比对显示,不同鱼类g型溶菌酶的氨基酸序列之间具有较高的保守性,系统进化树分析表明,该基因与草鱼(Ctenopharyngodon idella)g型溶菌酶亲缘关系较近。荧光定量PCR结果显示g型溶菌酶基因在团头鲂各组织中均有表达,肠道中表达量最高,同时在胁迫和恢复过程中该基因在肝和脑中的表达规律相似,均在胁迫时表达量上调,恢复后表达量相对下降,而g型溶菌酶在鳃中的表达有所不同,可推测g型溶菌酶基因参与了氨氮应激分子过程。

We cloned the c DNA encoding g-type lysozyme from blout snout bream（ Megahbrama amblycephala） by using rapid amplication of c DNA ends（ RACE） approach. The full length c DNA of g-type lysozyme was 719 bp,consisting of a 5＇ untranslated region of 71 bp,a 3＇ untranslated region of 90 bp and an open reading frame of 558 bp. Based on amino similarity comparison,the three catalytic sites and four substrate binding sites were highly conserved among different fishes,and phylogenetic tree analysis indicates that g-type lysozyme of M. amblycephala had close relationship with Ctenopharyngodon idella. Quantitative real-time PCR analysis shows that the g-type lysozyme gene was expressed in most tissues of M. amblycephala with highest expression in intestinal. After ammonia-N challenge and recovery,the relative expression levels of g-type lysozyme in liver and brain were higher than that in the control at 12 th,24th,48 thand 72ndhour of post-stress,followed by a gradual decrease at 72 thhour of recovery. By contrast,the relative expression level of g-type lysozyme in gill was lower than that in the control from 3rdhour to 72 ndhour of post-stress,but peaked at 72 ndhour of recovery. It is speculuted that g-type lysozyme gene involved in molecular processes of ammonia-N stress.