尼罗罗非鱼干扰素诱导蛋白44基因克隆及表达分析

Cloning and expression analysis of interferon induced protein 44 gene in Nile tilapia(Oreochromis niloticus)

【目的】探索干扰素诱导蛋白44基因(ifi44)在尼罗罗非鱼各组织的分布特征及其响应无乳链球菌感染和聚肌胞苷酸[Poly(I∶C)]刺激过程中的表达模式,为进一步揭示ifi44基因在鱼类免疫应答中的作用机制打下基础。【方法】克隆On-ifi44基因开放阅读框(ORF),通过ExPASy、TMHMM-2.0、Euk-mPLoc 2.0、SOPMA和SWISS-MODEL等在线软件进行生物信息学分析,根据单细胞转录组测序结果分析On-ifi44基因在细胞层面的分布情况,并采用实时荧光定量PCR检测On-ifi44基因在尼罗罗非鱼各组织中的表达分布特征及在无乳链球菌感染和Poly(I∶C)刺激后的时序表达情况。【结果】On-ifi44基因ORF序列全长1437 bp,编码478个氨基酸残基,其编码蛋白分子量为52.7 kD,理论等电点(pI)为4.97。On-ifi44含有1个TLDc_dom结构域(1~157 aa)和1个GTP-bd结构域(197~322 aa),不含跨膜结构域。On-ifi44氨基酸序列与奥利亚罗非鱼ifi44氨基酸序列的相似性为97.94%;基于ifi44氨基酸序列相似性构建的系统发育进化树也显示On-ifi44氨基酸序列与奥利亚罗罗非鱼ifi44氨基酸序列聚为一支,二者具有较近的亲缘关系。On-ifi44基因在尼罗罗非鱼的肠道、肝脏、鳃、皮肤、脾脏、体肾、胸腺、脑、头肾、肌肉、心脏等11个组织中均有表达,且主要在T细胞亚群表达;经无乳链球菌感染和Poly(I∶C)刺激后,On-ifi44基因在尼罗罗非鱼脾脏、肾脏、头肾和肠道组织中的相对表达量均发生变化,且存在明显的时间依赖性。【结论】On-ifi44含有1个TLDc_dom结构域和1个GTP-bd结构域,进化保守且在不同物种间具有相似的生物学功能;On-ifi44基因在无乳链球菌感染和Poly(I∶C)刺激后呈时间依赖性上调表达,表明ifi44基因作为重要的调节因子,参与了尼罗罗非鱼抗细菌感染、抗病毒增殖的免疫应答过程。

【Objective】The expression characteristics among different tissues and the expression pattern of interferon induced protein 44 gene(ifi44)after Streptococcus agalactiae infection and polyinosinic-polycytidylic acid[Poly(I∶C)]challenge were explored in present study to lay a theoretical basis for further researching the function and mechanism by ifi44 in the immune response of fish.【Method】The open reading frame(ORF)of On-ifi44 gene was cloned and its bioin-formatics was analyzed by ExPASy,TMHMM-2.0,Euk-mPLoc 2.0,SOPMA and SWISS-MODEL online softwares.The single-cell RNA-Seq(scRNA-seq)data were employed to analyze the distribution of On-ifi44 gene at the cellular level.The expression distribution of On-ifi44 gene in various tissues of healthy Nile tilapia and the sequential expression after S.agalactiae infection and Poly(I∶C)stimulation were detected by real-time fluorescent quantitative PCR.【Result】The open reading frame of the On-ifi44 gene was 1437 bp,encoding 478 amino acids residues with a protein molecular weight of 52.7 kD and theoretical isoelectric point(pI)of 4.97.On-ifi44 contained a TLDc_dom domain(1-157 aa)and a GTP-bd domain(197-322 aa),but no transmembrane domain.Amino acid sequence similarity between On-ifi44 and Oreochromis aureus ifi44 was 97.94%,phylogenetic tree based on amino acid sequence similarity of ifi44 amino acid sequence similarity showed that On-ifi44 amino acid sequence and O.aureus ifi44 amino acid sequence clustered into a single lineage,which meaned that they had a close genetic relationship.On-ifi44 gene was expressed in 11 tissues of Nile tilapia,including intestine,liver,gills,skin,spleen,kidney,thymus,brain,head-kidney,muscle and heart,and mainly expressed in the T cell subgroup.After S.agalactiae infection and Poly(I∶C)stimulation,the expression of On-ifi44 gene was changed significantly in the spleen,kidney,head-kidney,and intestinal,and with obvious time-dependence.【Conclusion】On-ifi44 contains one TLDc_dom domain and one GTP-bd domain,which is evolutionarily conserved and has similar biological functions among different species.The expression of On-ifi44 gene is up-regulated in a time-dependent manner after S.agalactiae infection and Poly(I∶C)stimulation,indicating that ifi44 gene,as an important regulatory factor,is involved in the immune response of Nile tilapia against bacterial infection and viral proliferation.