星斑川鲽MHC Ⅱ恒定链Ii基因的克隆和表达特性

cDNA cloning and expression characteristics of MHCⅡ invariant chain in Platichthys stellatus

为了研究星斑川鲽MHCⅡ类分子的作用及调控机制,实验通过SMART-RACE技术克隆得到了星斑川鲽MHCⅡ恒定链(MHCⅡIi)的全长cDNA序列,其长度为1766 bp,包含135 bp的5′非编码区、837 bp的开放阅读框和794 bp的3′非编码区。该基因共编码279个氨基酸。理论分子量为30.848 ku,等电点为6.89。与已知物种MHCⅡIi进行同源性比对,结果与狼鲈、紫红笛鲷和鳜关系较近,同源性均为79%。利用quantitative realtime PCR(qRT-PCR)技术检测了MHCⅡIi在星斑川鲽不同组织中的表达,以及爱德华氏菌感染前后对该基因在不同组织中表达水平的影响,结果显示:在脾脏、头肾、肝脏、后肠、性腺、心脏、血液、鳃和肌肉组织中,MHCⅡIi mRNA均有表达,但在表达量上有明显差异,脾脏和头肾组织相对表达水平较高,鳃、血液、肌肉、心脏和性腺中的表达水平较低。病原感染后,免疫相关组织脾脏和头肾的表达水平升高最明显,肝脏和后肠的表达水平也略有升高,但变化不明显。本研究可为星斑川鲽MHCⅡ类分子的作用机理提供理论依据,同时为海水养殖鱼类的抗病遗传育种工作提供研究基础。

In the present study, full length cDNA sequence of the major histocompatibility complexⅡ invariant chain（MHCⅡ） Ii gene was cloned in Platichthys stellatus by using 3′RACE and 5′RACE technique, and the sequence and structural analysis of the MHCⅡ Ii gene by using bioinformatics method. The results showed that the MHC Ⅱ Ii cDNA sequence consisted of 1766 bp, including 135 bp at 5′ region, 794 base pairs at 3′untranslated region and an open reading frame with 837 bp which could encode 279 amino acids. The theoretical molecular weight was 30.848 kDa, pI was 6.89. Comparison of the amino acid sequences and phylogenetic analysis showed that the P. stellatus MHC II Ii had the close relationship with Dicentrarchus labrax, Lutjanus argentimaculatus and Siniperca chuatsi. The results of different tissues indicated that the expression levels of MHCⅡ Ii mRNA were higher in spleen and head kidney, lower in gill, blood, muscle, heart and gonads by Real-time Quantitative PCR technique. After pathogen infection, the expression levels of immune spleen and head kidney increased most significantly, and liver and intestinal expression levels increased slightly. This study can provide a theoretical basis for the function and regulation mechanism of P. stellatus MHI, and for basic research of marine fish disease resistance breeding.