Evolutionary studies of genus Tribolodon based on major histocompatibility complex genes.

主要组织相容性复合体（MHC）是脊椎动物免疫防御系统的重要分子。在哺乳动物研究中，MHC基因通过正选择被认为是高度可变的。然而，MHC的进化研究并非如此。经常在硬骨鱼身上进行。在本研究中，我们建立了一种鲤科动物Tribolodon属的MHC基因分析体系，并基于MHC基因推断Tribolodon的进化特征。利用在山形县Mogami河捕获的hakont.s，我们试图从DNA中检测MHC I类基因。利用鲤鱼和斑马鱼ⅰ类基因设计的引物，成功扩增和克隆了hakont . MHCⅰ类基因。与鲤鱼和斑马鱼的序列比对，鉴定为ⅰ类α3。然而，hakonensis其他个体的部分序列无法被识别。这样的序列很可能是那些没有被表达的假基因。因此，很难从DNA中检测到I类基因。我们进一步尝试检测MHC II类基因的表达。利用mRNA合成的cDNA，扩增并克隆了hakonensis II类基因。检测到的hakont .hakonensis序列为IIB类基因。根据邻域连接法（NJ）构建了翻译后氨基酸序列的系统发育树。NJ树表明，hakonensis IIB类在4个位点上编码，每个位点上都存在等位基因。在该树中，一些T.hakonensis被包括在鲤鱼和斑马鱼的同一集群中，而其他的则形成独立的集群。这表明MHC IIB类基因在这种鲤形成物种之前已经复制了几次。因此，高可变MHC基因有望成为硬骨鱼进化研究的可用标记。

Major histocompatibility complex (MHC) is important molecules in immunodefense system in vertebrates. In mammalian studies, MHC genes are regarded hypervariable by positive selection. However, evolutionary studies of MHC are not. frequently carried out in teleosts. In this study, we undertook to establish analysis system of MHC genes of a cyprinid, genus Tribolodon and to deduce evolutionary features of Tribolodon based on MHC genes.Using T.hakonensis that were caught in the Mogami River, Yamagata, we tried to detect MHC class I genes from DNA.Using primers designed from class I genes of a carp and a zebrafish, we succeeded amplification and cloning of MHC class I genes of T.hakonensis. The aligned sequences with those of a carp and a zebrafish were identified as class I α3. However, some sequences of other individuals of T.hakonensis could not be identified. Such sequences are probably those of pseudogenes that are not expressed.Thus, it was difficult to detect class I genes from DNA.We further tried to detect MHC class II genes expressed. Using cDNA synthesized from mRNA, class II genes of T.hakonensis were amplified and cloned. The detected sequences of T.hakonensis were identified as class IIB genes. A phylogenetic tree of amino acid sequences translated was constructed according to neighbor joining (NJ) method. The NJ tree suggests that class IIB of T.hakonensis are encoded at four loci and that alleles exsist at each locus. In the tree, some of T.hakonensis are included in same clusters of a carp and a zebrafish, and others form independent own clusters. It suggests that MHC class IIB genes have been duplicated some times before such cyprinids were speciated. Thus, hypervariable MHC genes are expected as available markers for evolutionary studies also in teleosts.