Immune System And High-altitude Adaptation Study Of Wolf(Canis Lupus) Based On Blood Transcriptomic Analysis

The grey wolf(Canis lupus),one of the world's most widely distributed terrestrial mammals,has strong ability to adapt to multiple ecological niches.However,little is known about the genetic mechanism underlying the adaption to meta-habitat in wolf.Using methods of bioinformatics and molecular ecology on transctiptomic and related gene data,we elucidated the adaptive mechanism of wolf to meta-habitat from the perspective of immunity system,which has a crucial role for animals in adapting to changeable environment and expanding ecological niches.Additionally,comparative transcriptome analysis was employed to explore the adaptive mechanism of the Tibetan wolf to Tibetan Plateau.In a word,the objective of this study was to explore the adaptive mechanism underlying the adaptive to meta-habitat in wolf from two dimensions of immune generality and high-altitude adaptive peculiarity.We firstly established blood transcriptomic libraries of 8 wolves originated from different habitat types.After quality control,all the retained clean reads were assembled into unigenes based on genome-guided de novo transcriptomic assembly strategy.The assembled unigenes were then searched against 6 public databases and performed functional annotation.By combining unigene annotation results and expression information,we made a comprehensive analysis on wolf's immune system.Furthermore,MHC and TLR gene families,which play important roles in habitat adaption and parasite resistance for mammals,were selected to perform expression and molecular evolution analysis.Main results of this section were as follows:(1)In total,123,851 unigenes were obtained based on genome-guided de novo transcriptome assembly method,with an average length of 845 bp and N50 length of 1,121 bp.Re-mapping results showed that up to 83.7%reads were mapped to the unigenes,which indicating a high assembly quality of unigenes.(2)All the unigenes were then annotated to 6 databases to retrieve functional information.A total of 81,925(66.15%)unigenes were annotated to 6 databases,and 28,383(22.92%)unigenes were simultaneously annatated to all databases.According to GO classifications,24,663 unigenes were assigned to 13,016 GO terms of three domains including BP.CC and MF.Additionally,a considerable number of unigenes were assigned to GO categories associated with immune system process(1,721)and viral reproduction(374).KEGG pathway analysis showed that 7.682 unigenes were mapped to 335 pathways,and 969 unigenes were related to 15 immune signaling pathways,which including pathways involved by MHC and TLR genes.By KOG classifications,16,238 unigenes were annotated into 25 categories.(3)Based on the RSEM quantitation results,at least 7 MHC genes were detected in wolf blood transcriptome.Moreover,DLA-64 was expressed superiorly than DLA-79 with FPKM value was 371.For TLR genes,14 unigenes were identified for 9 TLR genes except for TLR6,and TLR9 was the most highly expressed TLR gene.Several highly expressed(FPKM top 0.1%)immune-related genes were also identified in wolf blood,such as LYZ,Fc?R ? and CD74.(4)Evidence for historical positive selection was detected on DLA-12 and DLA-88,a pattern expected for MHC under balancing selection.Screening for recombination yielded two DLA-88 alleles as intralocus recombinants and one DLA-64 allele and one DLA-79 allele as interlocus recombinants.Further analysis identified two significant breakpoints with the first breakpoint located the boundary of exon 2 and 3,supporting recombination through the mechanism of exon shuffling.Collectively,above findings suggested that positive selection and recombination shaped the evolutionary pattern in MHC I genes.Comparison of dN and ds revealed a distinct overall purifying selection for all TLRs across the carnivora lineage,and viral TLR suffered stronger purifying selection.Species-specific positive selection indicated more TLR and sites were under selection in wolf than in panda and ferret.Above results suggested that wolf may possess ability to sense more pathogenic bacteria for well adapted to meta-habitat.On the other hand,comparative transcriptome analysis was employed to investigate the differential expressed genes(DEGs)in peripheral blood between highland and lowland wolves,with the aim of illuminating the adaptive mechanism of the Tibetan wolf to the Tibetan Plateau.Using dog genome as reference,we totally obtained 87.8%mapped reads that covered 16,290 genes across all the 8 transcriptome samples.Several well known hypoxia-related genes,such as EPAS1,ANGPT1 and RYR2,were also detected in our transcriptome data.Main results of this section were as follows:(1)Comparisons of the transcriptional profiles between highland and lowland wolf groups revealed 90 DEGs when fold change>= 2 and adjusted p<0.05 were used as cutoff values.Further analysis revealed that 23 DEGs were up regulated in highland group.The qRT-PCR results were mostly consistent with the expression levels determined from the RNA-Seq data.(2)GO enrichment analysis demonstrated that the identified DEGs were enriched in specific biological processes with regard to ATP metabolic process,electron transport,and hypoxic response.KEGG pathway enrichment results showed that 11 pathways were significantly enriched(p<0.05),in which oxidative phosphorylation was the most significantly enriched(p = 0.00021)KEGG pathway.Above results indicated that changes in aerobic respiration and energy metabolism existed in Tibetan wolf gene expression profiles,and these adaptive changes may make Tibetan wolf well adapted to the Tibetan Plateau.(3)Further functional analysis on DEGs suggested that adaptive evolution occur within many physiological processes,which including energy metabolic,HIFs expression,ROS degradation,DNA repair and vascular system.This observation was consistant with previous genome-wide studies of high altitude adaptive mechanism on other mammals,and suggested convergent evolution may happened within many species native to the Tibetan Plateau to yield similar phenotypic adaptive responses.At last,we performed selection pressure analysis on mitochondrial proteins of Tibetan wolf for the vital role played by mitochondrion in aerobic respiration and energy metabolism.Phylogenetic analysis divided extant wolves into two clades with Tibetan wolves grouped to one clade,indicating a distinct phylogenetic position for Tibetan wolf.Selection pressure analysis revealed different selection pressure for ancestral mitochondrial protein sequences of Tibetan and lowland wolf.However,no signal of positive selection was detected in ancestral mitochondrial protein sequence of Tibetan wolf.In conculsion,our study presented the first insight into the characteristics of wolf immune system by RNA-Seq,with the emphasis of two important receptor-like gene families,i.e.MHC and TLR.Our study established the foundation for thoroughly understanding the role of immune system which played in adaptive evoluion to meta-habitat for wolf.Additionally,our study also demonstrated the adaptive mechanism of Tibetan wolf to Tibetan Plateau by genome-wide transcriptional analysis.The identified DEGs from blood transcriptome data were involved in many changed physiological processes,and may play key roles in high altitude adaption.These finding also provided clues and basis for us to further explore the gene expression changes through transcriptome data of other tissues.