团头鲂主要组织相容性复合体Ⅰ类基因全长cDNA的克隆及组织表达分析

Full length cDNA cloning and tissue expression of major histocompatibility complex (MHC) class Ⅰ from blunt snout bream (Megalobrama amblycephala)

为了解团头鲂MHCⅠ类基因的结构,采用cDNA末端快速扩增(Rapid-amplification of cDNA ends,RACE)技术,首次成功克隆了团头鲂"浦江Ⅰ号"F0基础群体及选育F8世代的MHCⅠ类基因,共获得3条cDNA全长序列。序列全长为2 040～2 079 bp,含有87～102 bp的5'-UTR,1 035～1 044 bp的编码区(包括信号肽,alpha 1、alpha 2和alpha 3三个结构域,跨膜区和胞质区)及911～946 bp的3'-UTR区,分别编码347、344个氨基酸。F8世代序列的核苷酸/氨基酸的同源性很高,为89.0%/93.0%,而与F0世代的同源性较低(75.3%～77.0%和71.1%～71.4%),呈现出明显的分子多态性。分析表明,团头鲂具有经典的MHCⅠ类分子的空间结构,与人类HLA-A2的抗原肽结合区的晶体结构相比,二者的差异主要集中在5个(I～V)区上。在Neighbor-joining(NJ)法构建的分子进化树中,团头鲂与草鱼的亲缘关系最近,与鲑鳟鱼类、爬行类、鸟类、哺乳类及人类的亲缘关系则渐远。RT-PCR组织表达分析表明,团头鲂MHCⅠ类基因在所检测的的8个组织中均表达,在鳃、头肾和血液中有较强的转录本,中等强度表达于肝脏和脾脏,在后肾、肠和肌肉中表达较弱。

RACE（Rapid Amplification of cDNA Ends） technique was used to clone ＂Pujiang No.1＂ blunt snout bream（Megalobrama amblycephala） MHC class Ⅰgene（Meam-MHC Ⅰ）,and total three full-length cDNA sequences in the F0 and F8 generations were obtained by cloning and sequencing method,respectively.The sequences were 2 040-2 079 bp in length,including an 87-102 bp 5′-UTR,a 1 035-1 044 bp coding region,containing signal region,three（α1,α2,α3） domains,a transmembrane and cytoplasmic region（TM/CY）,and 911-946 bp 3′-UTR.The coding region had 344-347 amino acids.Within F8 generation,the nucleotide/amino acid sequences similarities were as high as 89.0%/93.0%.But the values were relatively as low as 75%-77% /71% between generations,indicating obviously sequence polymorphism.The Meam-MHC I exhibited a classical 3D molecular structure,and compared to the PBR region of HLA-A2,the major differences were mainly in the five regions（I-V）.It was revealed in the neighbor-joining tree that the blunt snout bream had the closest relationship with grass carp,far from salmon and trout,and still farther from,chicken and human.Semi-quantitative RT-PCR analysis demonstrated that Meam-MHC I transcript was detected at high level in gill,head kidney and blood,moderate in liver and spleen,and low in trunk kidney,gut and muscle.