尼罗罗非鱼MHCⅠa全长cDNA的克隆、多态性及组织表达特征

cDNA isolation, polymorphism analysis, and tissue expression of MHC Ⅰa of Nile tilapia(Oreochromis niloticus)

通过同源克隆和末端快速扩增（RACE）方法,获得尼罗罗非鱼（Oreochromis niloticus）的MHC ？α全长cDNA,分析其多态性和组织表达特征。获得的尼罗罗非鱼MHC？αcDNA全长为1533 bp,其ORF为1053 bp。编码的蛋白质分子包含信号肽、MHC结构域、IGc1结构域和跨膜区,并存在丰富的磷酸化位点。与其他硬骨鱼类和哺乳类的相似性在26.86%~74.0%。从6尾鱼中共分离得到6条不同的MHC ？α cDNA序列,其序列中存在多个单核苷酸多态性位点,说明MHC？αcDNA存在丰富的多态性。qPCR检测表明, MHC？α基因在脾和心中表达量较高,中等程度表达于鳃、肠和肾中,在脑、肝、胃、皮肤和肌肉中表达量较低。对尼罗罗非鱼进行人工感染无乳链球菌（Streptococcus agalactiae）后, MHC？α基因的mRNA表达水平在鳃、肾、心脏和脾4个组织中均发生了不同程度的变化。结果表明MHC Iα基因参与罗非鱼免疫反应,在免疫应答中发挥重要功能。本研究通过研究尼罗罗非鱼MHC？α的生物学活性、功能和表达调控,旨在为揭示尼罗罗非鱼免疫抗感染机制提供基础资料,并为利用MHC？α作为候选基因筛选尼罗罗非鱼抗病相关分子标记、辅助抗病品系的选育奠定基础。

Major histocompatibility complex is a genetic region of the chromosome. It consists of a series of closely linked highly polymorphic loci that stimulate the body’s immune response by encoding a class of cell sur-face transmembrane protein that binds to T lymphocytes and presents endogenous and exogenous antigens to it. Based on the techniques of homology cloning and RACE-PCR, full-length cDNA of MHC ？α was cloned from Nile tilapia. In addition, cDNA polymorphism and mRNA expression were also analyzed. The total length of MHC1α cDNA was 1 553 bp, which the ORF （open reading frame） was 1 053 bp in length. Sequence analysis showed that the deduced protein sequence of MHC？αincluded a leader peptide , a MHC-I domain, an IGc1domain, and a transmembrane region. The putative protein had 26.86%–74.0% identity with those of other teleosts and mammals. Six different MHC？αcDNA sequences were obtained from six individuals. There were multiple single nucleotide polymorphisms among the six MHC？αcDNA sequences. This indicates that MHCα？cDNA has abu n-dant polymorphism. qPCR detected the highest expression level of MHCα？in spleen and heart , moderate expres-sion in gill, kidney and intestine, and the lowest expression in brain, liver, stomach, skin and muscle. Different changes were found in the expressions of MHαC ？mRNA in the gill , kidney, heart and spleen after being chal-lenged with Streptococcus agalactiae. The findings provide useful information for further investigation to select a disease resistant molecular marker of Nile tilapia using MHC Iαas a candidate gene.