尼罗罗非鱼Cullin-1基因克隆及表达

Cloning and Tissue Expression Analysis of the Cullin-1 in Nile Tilapia(Oreochromis niloticus)

【目的】探究尼罗罗非鱼(Oreochromis niloticus)Cullin-1(CUL1)基因在尼罗罗非鱼抵抗病原细菌过程中的作用。【方法】从尼罗罗非鱼脾脏组织中克隆Cullin-1基因(记为On-CUL1),对其进行生物信息学分析,应用实时荧光定量PCR技术分析On-CUL1在健康鱼胸腺、鳃、肝、脾、肠道、头肾、脑、肌肉、皮肤、血液组织,以及经无乳链球菌刺激后病鱼脾、肠道、头肾中的表达。【结果与结论】On-CUL1(GenBank登录号XM003459470.5)编码区为2 331 bp,编码776个氨基酸,理论分子质量为89 ku,理论等电点为8.16。氨基酸序列分析显示,On-CUL1有1个高度保守的泛素样蛋白结构域CUL1 Nedd8(ubiquitin-like domain),N′端有一个的信号肽序列。系统进化树分析表明,On-CUL1序列与伯氏拟丽鱼(Haplochromisburtoni)CUL1相似性最高,为98.7%。q RT-PCR显示CUL1在健康尼罗罗非鱼各组织中均有表达,其中在头肾中表达量最高,其次是胸腺、脑、皮肤,在肠道中表达量最低。经无乳链球菌刺激后,On-CUL1表达量在脾脏和肠道中均显著上调,头肾、脾脏和肠道均在48h表达量最大。CUL1参与尼罗罗非鱼抵抗无乳链球菌过程中的免疫应答。

【Objective】To explore the role of cullin-1(CUL1)gene of the Nile tilapia(Oreochromis niloticus)in the defense of pathogenic bacteria.【Method】The CUL1 gene(named On-CUL1)was cloned from the spleen of the Nile tilapia(Oreochromis niloticus),and bioinformatics analysis of the gene was performed,and the expression of On-CUL1 in the thymus,gills,liver,spleen,intestine,head kidney,brain,muscle,skin,and blood tissues of healthy fish,and the spleen,intestine,and head kidney of Streptococcus alactis stimulated fish was analyzed by fluorescence quantitative PCR.【Result and Conclusion】The coding region of On-CUL1 gene was 2331 bp,and encoding a 776 amino acids protein with a theoretical molecular mass 89 ku,and isoelectric point 8.16.On-CUL1 had a highly conserved ubiquitin-like domain,CUL1 Nedd8(ubiquitin-like domain),and a signal peptide sequence at the N terminal.On-CUL1 showed the highest similarity with Haplochromis burtoni(98.7%).On-CUL1 was expressed in all tissues of healthy Nile tilapia,with the highest expression in head kidney,followed by thymus,brain and skin,and the lowest expression in intestine.After it was stimulated by inactivated S.agalactiae.The expression of On-CUL1 was significantly up-regulated in the spleen and intestine.The expression levels in the body kidney,spleen and intestine peaked at 48 h.【Conclusion】On-CUL1 was involved in the immune response of Nile tilapia against S.agalactis.