长丰鲫c型溶菌酶基因的克隆及其在细菌感染条件下的表达分析

为了解c型溶菌酶基因与长丰鲫(Chang Feng Carassius auratus)的抗菌效应关系,本研究利用同源克隆方法获得长丰鲫c型溶菌酶基因cDNA全长序列。结果显示:c型溶菌酶基因全长698 bp,包括5'端非翻译区60 bp,3'端非翻译区200 bp,开放阅读框438 bp,编码145个氨基酸。长丰鲫c型溶菌酶基因在肾脏组织中表达量最大,在脾脏、肠道、心脏和脑中大量表达,在肝脏和鳃中表达量相对较低,在皮肤和肌肉中几乎不表达。长丰鲫在感染迟钝爱德华氏菌、嗜水气单胞菌和金黄色葡萄球菌后,在肝脏、肾脏、脾脏和鳃等组织中基因表达量均发生显著变化,出现不同程度的上调。感染迟钝爱德华氏菌后,在脾脏中的上调幅度最大,其次为鳃、肝脏和肾脏;感染嗜水气单胞菌后,在脾脏中上调程度最大,其次是鳃;感染金黄色葡萄球菌后,在脾脏中上调幅度最大。在肝脏和肾脏中,经金黄色葡萄球菌刺激后c型溶菌酶基因的上调幅度最高;在脾脏和鳃中,经嗜水气单胞菌感染后c型溶菌酶基因上调幅度最高。三种刺激后,c型溶菌酶基因升高幅度的不同,说明不同刺激使鱼体组织产生的应激反应能力不同。

Molecular cloning and expression analysis of c-type lysozyme gene inChangFeng Carassius auratus during bacterial infection

To understand the biological role of c-type lysozyme in the ChangFeng C.auratus,in this study,c-type lysozyme is cloned by homology cloning strategy from ChangFeng Carassius auratus.The results showed that the full length cDNA sequence of c-type lysozyme is 698 bp,including a 438 bp open reading frame with a coding potential for a 145-amino acid protein,a 60 bp 5′-untranslated region and a 200 bp 3′-untranslated region.c-type lysozyme mRNA was expressed ubiquitously in the tissues examined and abundantly expressed in kidney,It had a large number of expression In the spleen,intestine,heart and brain,but in the liver and gills the expression was relatively low,almost no expression in the skin and muscle.After intraperitoneal injection with Edwardsiella tarda,Aeromonas hydrophila and Staphylococcus aureus respectively,the mRNA levelsofc-typelysozyme was more or less rapidly upregulated in liver,kidney,spleen and gill.After intraperitoneal injection with Edwardsiella tarda,higher degree of the mRNA levels of c-type lysozyme was largest in spleen,followed by gills,liver and kidney;After intraperitoneal injection with Aeromonas hydrophila,escalation of the mRNA levels of c-type lysozyme was largest in spleen,followed by gills;After intraperitoneal injection with Staphylococcus aureus,up-regulation of the mRNA levels of c-type lysozyme was largest in spleen.In liver and kidney,the uptake rate of the c-type lysozyme gene was the highest when stimulated by Staphylococcus aureus.But in spleen and gill,the up-scaling of the mRNA levels of c-type lysozyme gene was the highest when stimulated by Aeromonas hydrophila.After the stimulation of three bacteria,higher degree of the mRNA levelsofc-typelysozyme was different,It showed different stimulus may make different stress reaction ability in the fish tissue.