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中的重要分子。在哺乳动物研究中，MHC基因被认为是高变的积极选择。然而，MHC的进化研究却不是这样。经常在硬骨鱼中进行。本研究以日本山形县最上川捕获的鱼（Tribolodon）为研究对象，尝试从DNA中检测鱼MHC Ⅰ类基因，并根据鲤和斑马鱼的MHC Ⅰ类基因设计引物，成功地扩增和克隆了鱼MHC Ⅰ类基因。与鲤鱼和斑马鱼的序列比对，鉴定为I类α3。然而，一些序列的其他个人的T.hakonensis无法确定。这些序列可能是不表达的假基因。因此，很难从DNA中检测I类基因。我们进一步尝试检测表达的MHC II类基因。利用从mRNA合成的cDNA，扩增并克隆了T.hakonensis的II类基因。检测到的序列被鉴定为IIB类基因。根据邻接法（NJ）构建了翻译的氨基酸序列的系统发育树。NJ进化树表明，Hakonensis的IIB类基因由4个位点编码，每个位点都存在等位基因。在该树中，一些T.hakonensis被包括在鲤鱼和斑马鱼的相同集群中，而另一些则形成独立的集群。这表明，MHC Ⅱ B类基因已经复制了一些时间之前，这样的鲤科鱼类的物种。因此，高变MHC基因有望成为硬骨鱼类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.