Liu Guangshuai, Zhang Honghai, Sun Guolei, Zhao Chao, Shang Shuai, Gao Xiaodong, Xia Tian, Yang Xiufeng
Qufu Normal University, Jingxuan Street No. 57, Qufu, Shandong province, China.
BMC Genomics. 2017 Aug 7;18(1):584. doi: 10.1186/s12864-017-3983-0.
The wolf (Canis lupus) is one of the most widely distributed terrestrial mammals, because it is well adapted to various ecological niches and their corresponding pathogen environments. Immunological competence is a crucial factor involved in adapting to a changing environment and fighting pathogen infection in animals. In this study, the peripheral blood transcriptome of wolves was generated via RNA-seq to advance understanding of the wolf immunome, with a special focus on the major histocompatibility complex class I (MHC I) and toll-like receptor (TLR) gene families, which are involved in pathogen recognition and defense.
The blood transcriptomic libraries of eight wolves originating from Tibet and Inner Mongolia were sequenced, and approximately 383 million reads were generated. Using a genome-guided assembly strategy, we obtained 123,851 unigenes, with a mean length of 845 bp and an N50 length of 1121 bp. On the basis of BLAST searches against the NCBI non-redundant protein database (Nr), a total of 36,192 (29.22%) unigenes were annotated. For functional classification, 24,663 unigenes were assigned to 13,016 Gene Ontology (GO) terms belonging to 51 sub-categories of the three main GO categories. Additionally, 7682 unigenes were classified into 6 Kyoto Encyclopedia of Genes and Genomes (KEGG) categories, in which the most represented functional sub-categories were signal transduction and the immune system, and 16,238 unigenes were functionally classified into 25 Eukaryotic Orthologous Groups (KOG) categories. We observed an overall higher ω (d /d ) value at antigen-binding sites (ABSs) than at non-ABS regions as well as clear evidence of intergenic/intragenic recombination events at wolf MHC I loci. Additionally, our analysis revealed that carnivorous TLRs were dominated by purifying selection, with mean ω values at each TLR locus ranging from 0.173 to 0.527. However, we also found significant instances of positive selection that acted on several codons in pathogen recognition domains and were linked to species-specific differences in pathogen recognition.
This study represents the first attempt to characterize the blood transcriptome of the wolf and to highlight the value of investigating the immune system. Balancing selection and recombination have contributed to the historical evolution of wolf MHC I genes. Moreover, TLRs in carnivores have undergone adaptive evolution against the background of purifying selection, and a high level of adaptive evolution was detected in the wolf TLR system.
狼（Canis lupus）是分布最广泛的陆生哺乳动物之一，因为它能很好地适应各种生态位及其相应的病原体环境。免疫能力是动物适应不断变化的环境和抵抗病原体感染的关键因素。在本研究中，通过RNA测序生成了狼的外周血转录组，以增进对狼免疫组的了解，特别关注参与病原体识别和防御的主要组织相容性复合体I类（MHC I）和Toll样受体（TLR）基因家族。
对来自西藏和内蒙古的8只狼的血液转录组文库进行了测序，共产生了约3.83亿条 reads。使用基因组引导的组装策略，我们获得了123,851个单基因，平均长度为845 bp，N50长度为1121 bp。基于对NCBI非冗余蛋白质数据库（Nr）的BLAST搜索，总共注释了36,192个（29.22%）单基因。在功能分类方面，24,663个单基因被分配到13,016个基因本体（GO）术语中，这些术语属于三个主要GO类别的51个子类别。此外，7682个单基因被分类到6个京都基因与基因组百科全书（KEGG）类别中，其中最具代表性的功能子类别是信号转导和免疫系统，16,238个单基因在功能上被分类到25个真核直系同源组（KOG）类别中。我们观察到抗原结合位点（ABSs）处的ω（dN/dS）值总体上高于非ABS区域，并且在狼MHC I基因座处有明显的基因间/基因内重组事件的证据。此外，我们的分析表明，食肉动物的TLR主要受到纯化选择的影响，每个TLR基因座的平均ω值在0.173至0.527之间。然而，我们也发现了正向选择的显著实例，这些实例作用于病原体识别域中的几个密码子，并与病原体识别中的物种特异性差异有关。
本研究首次尝试对狼的血液转录组进行表征，并强调了研究免疫系统的价值。平衡选择和重组促成了狼MHC I基因的历史进化。此外，食肉动物的TLR在纯化选择的背景下经历了适应性进化，并且在狼TLR系统中检测到了高水平的适应性进化。
