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基因被认为是正向选择的高变量。然而，对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，我们致力于建立鲤科、斑马鱼属MHC基因的分析体系，并根据MHC基因推断出其进化特征。本研究以山形莫加米河捕获的日本血吸虫为材料，从DNA中检测MHC-I类基因，并利用鲤鱼和斑马鱼的MHC-I类基因设计引物，成功地扩增和克隆了日本血吸虫MHC-I类基因。与鲤鱼和斑马鱼的比对序列被鉴定为I类α3，而哈康锥虫其他个体的一些序列不能被鉴定。这样的序列很可能是假基因中没有表达的序列，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。利用合成的基因片段，扩增并克隆了日本血吸虫的第II类基因。哈康毛滴虫的检测序列被鉴定为IIB类基因。根据邻接法(Neighbor Join，NJ)构建了氨基酸序列的系统发育树。NJ树表明，哈空毛虫IIB类在4个座位上编码，每个座位上都存在等位基因。在这棵树中，一些斑马鱼被归入同一群鲤鱼和斑马鱼中，而另一些则形成了独立的自己的群。这表明，在这种鲤科动物形成之前，MHC类IIB基因已经复制了一些次。因此，高可变的MHC基因有望成为进化研究的有效标记，硬骨鱼也是如此。

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.