尼罗罗非鱼pik3r1基因的克隆和mRNA表达分析

Cloning Identification and mRNA Expression Analysis of pik3r1 Gene in Oreochromis niloticus

【目的】哺乳动物pik3r1基因参与多种免疫途径,探索pik3r1基因在罗非鱼(Oreochromis)中的作用。【方法】克隆尼罗罗非鱼(Oreochromis niloticus)pik3r1(命名为On-pik3r1)cDNA全长,对该基因进行生物信息学分析,并运用荧光定量PCR方法分析无乳链球菌(Streptococcus agalactiae)刺激后On-pik3r1 mRNA在各组织种的表达模式。【结果与结论】On-pik3r1基因编码区2190 bp,编码729个氨基酸,5′端非编码区(UTR)为542 bp,3′UTR为2248 bp,理论分子质量为83.99 ku,等电点为5.73。On-pik3r1与斑马拟丽鱼(Maylandia zebra)相似性最高(98.53%),与其他物种的同源性在70%以上,表明pik3r1在物种进化过程中高度保守。On-pik3r1在健康尼罗罗非鱼各组织中均有表达,在肌肉中表达量最高,其次是鳃、皮肤,在胸腺中表达量最低。经灭活无乳链球菌刺激后,On-pik3r1表达量在肠道、鳃、脾脏、头肾、脑部等5个组织中均极显著下调(P<0.01),在胸腺中表现为4 h时极显著下调(P<0.01),24、48、72 h时为显著下调(P<0.05)。On-pik3r1参与了罗非鱼的对无乳链球菌的免疫应答过程。

【Objective】To explore the role of Pik3r1 gene in Oreochromis,which has been shown to be involved in a variety of immune pathways in mammalian.【Method】The full length of the cDNA of the Nile tilapia(Oreochromis niloticus)pik3r1(named on-pik3r1)was cloned and identified.Also,bioinformatics analysis of the gene was carried out,and the expression patterns of on-pik3r1 mRNA in various tissues after stimulation by Streptococcus agalactis were studied by fluorescence quantitative PCR.【Results and Conclusion】The results showed that the CDS region of On-pik3r1 gene was 2190 bp,encoding 729 amino acids,5′UTR was 542 bp,3′UTR was 2248 bp,a theoretical molecular mass was 83.99 ku,and an isoelectric point was 5.73.On-pik3r1 has the highest similarity with Maylandia zebra(98.53%)and more than 70%homology with other species,which indicates that pik3r1 is highly conserved during species evolution.On-pik3r1 is expressed in all tissues of healthy Nile tilapia,the highest expression is found in the muscle,followed by gill,skin,and the lowest expression is found in thymus.After it was stimulated by inactivated S.agalactiae,the expression of On-pik3r1 was extremely down-regulated in the intestine,gills,spleen,head kidney,and brain.The expression in the thymus was extremely down-regulated at 4 h,and it was significantly down-regulated at 24 h,48 h and 72 h,.In conclusion,the results suggest that On-pik3r1 participates in the antibacterial immune response of tilapia.It lays a certain foundation for further research on the role of On-pik3r1 in the process of tilapia infection with S.agalactis.