凹耳蛙MHCII类B基因第二外显子多态性分析

两栖类正经历全球范围内的种群衰退,很多两栖动物集群灭绝事件与环境病原体(如壶菌(Batrachochytrium dendrobatidis)的侵扰有关。MHC基因的表达产物在有颌脊椎动物免疫应答过程中起关键作用,其多态性通常与动物对疾病的抗性或易感性密切相关,因而被认为是研究动物适应性进化的最佳候选基因之一。本文对中国特有的无尾两栖动物凹耳蛙(Odorrana tormota)MHC II类B基因多态性进行初步研究。首先,利用1对通用引物扩增出凹耳蛙MHC II类B基因exon2长约180bp的DNA片段。在此基础上,利用ligation-mediated PCR进一步获取侧翼未知序列,序列拼接后长2,030bp,包含exon2以及intron1和intron2的部分序列。基于上述序列设计出凹耳蛙B基因exon2特异性引物(IIQ1BU/IIQ1BD),对该物种黄山种群32个样品进行PCR扩增和克隆测序,共获得34个不同的等位基因,等位基因序列核苷酸和氨基酸变异位点的比例分别为16.17%(33/204)和26.87%(18/67),大多数氨基酸变异位点位于推测的抗原结合位点(antigen binding sites,ABS)。每个样品包含2-5个等位基因,结合等位基因序列特征以及cDNA表达分析结果,推测凹耳蛙至少拥有3个可表达的B基因座位。与文献报道的蛙科其他物种比较后发现,尽管凹耳蛙目前的分布区非常狭窄,但其MHC II类B基因多态性明显高于蛙科其他动物。等位基因碱基替换模式提示凹耳蛙MHC II类B基因曾经历过强烈的正选择作用,ABS区的dN值显著大于dS(P<0.05),PAML软件包CODEML程序中不同模型的似然比检测(likelihood rate test)结果同样支持上述推论,贝叶斯经验贝叶斯路径(Bayesian Em-pirical Bayes)共检测出5个显著受正选择作用的氨基酸位点。贝叶斯系统树的拓扑结构显示,无尾两栖类不同科的等位基因分别形成单系群,但蛙科不同属的等位基因未能形成单系群,蛙属绿池蛙(Rana clamitans)的1个等位基因与臭蛙属凹耳蛙的部分等位基因享有共同的谱系关系,提示蛙科不同属间的B基因存在跨种多态性。

Polymorphism of exon 2 of MHC Class Ⅱ B gene in the Chinese concave-eared torrent frog (Odorrana tormota)

Currently,amphibians are experiencing global population decline,and it is believed that several amphibian mass extinction events were caused by environmental pathogens(such as chytrid fungus).Major histocompatibility complex(MHC) genes play a critical role in the course of immune response in all jawed vertebrates.MHC gene is considered to be one of the best candidates to analyze animal’s adaptive evolution because its polymorphisms are usually associated with resistance or susceptibility to animal diseases.Here,we report our preliminary research on the allelic diversity of MHC class II B gene from the Chinese concave-eared frog(Odorrana tormota),a species endemic to eastern China.We initially amplified a 180-bp fragment of MHC II exon2 gene in O.tormota using published polymerase chain reaction primers.Based on these results,we successfully obtained sequences of the gene’s flanking regions using a ligation-mediated PCR method.After splicing,we obtained a sequence with length of 2,030 bp including whole exon2 and partial sequences of intron1 and intron2.Then two exon2-specific primers(IIQ1BU/IIQ1BD) were designed forthe species and were used to investigate the B gene diversity of a wild population(Huangshan Mt.,n=32) using PCR,cloning and sequencing.In total,34 distinct alleles were obtained and 2 to 5 alleles were found per individual.The proportion of variable sites for nucleotide and amino acid sequences across the 34 alleles was 16.17%(33/204) and 26.87%(18/67),respectively,and the majority of variable amino acids were located in antigen binding sites(ABS).Based on cDNA data and individual allelic diversity,we conclude that O.tormota possesses at least three class II B loci.These results showed that though the species exhibits a restricted distribution,the Chinese concave-eared frog displays high diversity at the B loci compared to that of other species in Ranidae.Patterns of nucleotide substitution exhibited the signature of positive selection.For example,in the ABS,dN was significantly greater than dS(P <0.05).This pattern was also echoed by results of a likelihood ratio test,and five amino acid sites under significant positive selection were detected by Bayesian analyses.Finally,topology of a Bayesian phylogenetic tree showed that alleles from different anuran families clustered into unique monophyletic groups,but that alleles from different genera within Ranidae did not form monophyletic groups.For example,an allele of Rana clamitans shared a common lineage with alleles of O.tormota.These results suggested that trans-species evolution may be a mechanism that maintains allelic polymorphism of the MHC II B gene across genera in the family Ranidae.