尼罗罗非鱼Xbp1-S基因克隆及表达分析

Molecular Cloning and Expression Analysis of Xbp1-S of Nile Tilapia(Oreochromis niloticus)

为了了解尼罗罗非鱼(Oreochromis niloticus)细胞转录因子Xbp1-S(On Xbp1-S)的基因序列特征及其在无乳链球菌(Streptococcus alactolyticus)应激和在B细胞分化中的作用,应用RACE克隆技术获得的On Xbp1-S基因全长1380 bp,包括开放阅读框ORF为1155 bp,5?端非编码区(5?UTR)长127 bp,3?端非编码区(3'UTR)长98 bp。On Xbp1-S序列分析推测该基因编码384个氨基酸,分子量为41.32 k Da,理论等电点为4.36。同源性分析显示,On Xbp1-S基因与其他鱼类的聚为一支,其中,与南极鳕(Notothenia coriiceps)相似性最高。荧光定量PCR及Western-blot结果显示,On Xbp1-S在各组织中均有表达,m RNA水平上在肝脏中表达量最高,蛋白水平上在胸腺中表达量最高,而在肌肉中表达量最低。无乳链球菌应激后,On Xbp1-S基因在肝脏和脾脏中的表达趋势相似,均在应激期间出现表达量上调,在192 h出现峰值。另外,免疫组化分析发现,On Xbp1-S因子在不同分化程度B细胞亚类中的表达呈现差异,在成熟B细胞中呈现高表达,而在未成熟B细胞中几乎不表达。研究结果表明,On Xbp1-S参与尼罗罗非鱼对无乳链球菌的免疫防御,和在B细胞分化中起作用。本研究将为进一步研究On Xbp1-S因子应答病原菌侵染的机理及促进B细胞分化机制提供理论依据。

X-box binding protein 1-S(Xbp1-S)is a key transcript factor mainly associated with B cell development and differentiation.The 1380 bp full-length cDNA of OnXbp1-S including 1155 bp ORF,127 bp 5'UTR and 98 bp 3'UTR was cloned with RACE,which encodes 384 amino acids with a molecular weight of 41.32 kDa and a theoretical isoelectric point of 4.36.Homology analysis revealed that the OnXbp1-S gene was homologous to other fish with the most similarity with Notothenia coriiceps.OnXbp1-S expresses in all tested tissues,with the highest mRNA expression in liver,and the highest protein level in thymus and the lowest protein level in muscle.Streptococcus agalactiae stimulation induced the expression of OnXbp1-S with the highest level at 192 h post-infection.In addition,mature B cells have a much higher OnXbp1-S level than naive B cells.Taken together,the results indicated that OnXbp1-S might be involved in the immune response in Nile tilapia against S.agalactiae infection,and play a role in B cell development and differentiation.