| Oxygen is necessary for aerobic organisms.Animals have evolved various oxygen sensing and response mechanisms to adapt to different hypoxic environments.High altitude hypoxia and subterranean hypoxia are two typical types of hypoxia on land.To compare the effects of two hypoxic environments on animal adaptation and evolution,three species of the field vole subfamily(Arvicolinae),Mandarin voles(Lasiopodomys mandarinus),Brandt's voles(L.brandtii)and Qinghai voles(Neodon fuscus)were selected for comparative study in this thesis.Among them,L.mandarinus belongs to subterranean rodents while L.brandtii and N.fuscus are both the ground mice at different altitude.To reveal the adaptive evolutionary mechanism in L.mandarinus and N.fuscus,we performed comparative transcriptomic study by analyzing the gene structure of unigenes.Specifically,the differential gene expression(DEGs)analysis relying on the reference transcriptome was used to study the hypoxic adaptation mechanism of the brain tissues in L.mandarinus and L.brandtii,which were treated under the conditions of acute hypoxia or chronic hypoxia in a closed oxygen chamber with normal pressure and then collected.Furthermore,we did transcriptome trend expression analysis to uncover the molecular mechanism of hypoxia adaptation in N.fuscus and L.brandtii by mimicking different hypoxia treatment with four types of altitude in a low-pressure oxygen chamber.This study systematically reveal the mechanisms of hypoxic adaptation and evolution in subterranean rodents and plateau mouse from the different aspects of transcriptome evolution and gene differential expression,providing new evidences for the theory of hypoxic adaptation and evolution in terrestrial mammals,and also new clues for the diagnosis and treatment of human hypoxic diseases.The major results are as follows:1.Brain transcriptome sequencing,unigenes assembly and annotationA total of 210 Gbp high-quality transcriptome data were obtained from 15 brain tissue samples in three voles.The length of unigenes N50 in these three species is greater than 2,300 bp and the transcript assembly shows good enough by BUSCO evaluation.Totally,there are 27572,27663 and 2 8314 unigenes annotated in N.fuscus,L.mandarinus and L.brandtii respectively.2.Evolutionary analysis of brain transcriptomes in three volesTaking N.fuscus,L.mandarinus and L.brandtii as the research objectives,Homo sapiens and Mus musculus as the outer species,we used the software Ortho MCL to filter out the homologous gene sequences by comparing the protein sequences.A total of 7039 single copy ortholog genes were screened out.The evolution rate(dN/dS)of the five species was significantly different(rank sum test,P<0.0001),and the difference between the species pairwise reached a very significant level(P<0.0001).N.fuscus has the fastest evolution rate which is 0.3022,followed by L.mandarinus and L.brandtii with 0.2862 and 0.2713,respectively.M.musculus and H.sapiens with 0.1933 and 0.1702 rank the last two.A total of 196 positive selection genes were identified in L.mandarinus,mainly enriched in peroxisomes(ko04146),DNA damage repair process(GO:0006281),etc.On the other hand,284 positive selection genes in N.fuscus were identified,which belong to not only those items of Oxidase bodies or DNA damage repair process same to L.mandarinus but also purine metabolism(ko00230),apoptosis process(GO:0006915)and redox process(GO:0055114),etc.3.Differential expression analysis of the brain transcriptome under severe hypoxic in L.mandarinus and L.brandtii1)The results of analysis with reference transcriptome showed that,under 5%acute hypoxic treatment,?A total of 460 DEGs were obtained from the L.mandarinus,of which up-regulated DEGs were mainly in development(GO:0032502)and response to stress(GO:0006950),and down-regulated DEGs were mainly in gene expression regulation and metabolic processes(GO:0051252,GO:0010468,GO:0019222);Significantly enriched pathways by KEGG(ko04510,ko04151,ko04015,ko04310)mainly were related to inhibiting apoptosis,promoting cell proliferation and angiogenesis;?A total of 1,120 DEGs were obtained from L.Among them,the function of up-regulated DEGs enrichment were mostly related to immune process(GO:0002376,GO:0006955),response to stimulus(GO:0009605,GO:0050896),locomotion(GO:0040011),etc.The down-regulated DEGs were mainly related to the regulation of cell surface receptor signaling pathway(GO:0007166).2)The results of GSEA enrichment analysis showed that ? L.mandarinus mainly up-regulated the functions of cell proliferation,angiogenesis and cell response to external stimuli,and down-regulated the processes of oxygen consumption such as oxidative phosphorylation,protein secretion and cell cycle;? The up-regulated gene set of L,brandtii has the same effect as the up-regulated gene set in L.mandarinus.The difference is that down-regulates genes of L.brandtti were also related to the DNA repairing pathway.4.Differential expression analysis of the brain transcriptome under chronic hypoxia in L.mandarinus and L.brandtii1)Transcriptome analysis results with reference genomes showed that,under 10%chronic hypoxic conditions,? A total of 439 DEGs were obtained in L.mandarinus,mainly for activation of HIF,focal adhesions,PI3K-Akt and Rap1 signaling pathways;?A total of 215 DEGs were obtained in L.which mainly belong to up-regulated neurotransmitter excitability regulation(GO:0005509)and immune(ko04610,ko04512,ko04060)related pathways.For the down-regulated DEGs,the related items mainly include the response to hypoxic stimulation(GO:0051716)and signal transduction of various types of membranes in vivo(GO:0023052,GO:0007165).2)The results of GSEA enrichment analysis show that ? The up-regulated gene set of L.mandarinus is consistent with the KEGG enriched one,both contain focal adhesion signaling pathway and natural killer cell-mediated cytotoxic pathway.The down-regulated gene set include many gene sets for neuro diseases such as Parkinson's disease,Huntington's disease or Alzheimer's disease,and also related RNA degradation processes.? The function of the up-regulated gene set of L.brandtii is mainly to regulate glycolysis and promote angiogenesis.The down-regulated gene set is not only consistent with the neurological disease pathway down-regulated in L.mandarinus,but also involves cancer pathways.5.Brain transcrip tome analysis of N.fuscus and L.brandtii upon high altitude hypoxiaFour simulated altitude treatment groups of 100 m,3Km,5Km,and 7Km were set,and DEGs were screened with |log2(fold change)|>2,P-adj<0.05.The number of DEGs in two voles increases with increased altitudes.According to the trend analysis,the two most significant trend blocks were selected in N.fuscus compared to L.brandtii,namely the linear up-regulation group and the linear down-regulation group.Of which,linear up-regulation groups in both two voles involve angiogenesis function(GO:0001525).Besides,the cell cycle(GO:0007049),protein transport(GO:0015031,GO:0006810),and the cell 's response to DNA damage stimulation(GO:0006974)were enriched in N.fuscus while response to hypoxia(GO:0001666)and cold(GO:0009409),glycolysis process(GO:0061621),and metabolic-related pathways(ko00010,ko01200)could be enriched in L.brandtii.Among the genes in the linear down-regulation group,N.fuscus had functional enrichment in terms of immune response and virus defense,while L.brandtii did not significantly enrich any functional pathway upon hypoxia.The major conclusions are as follows:1)N.fuscus,L.mandarinus and L.brandtii have relatively fast evolution rates(dN/dS),which should be a good biological model for comparative transcriptomics evolution analysis.Both of L.mandarinus and N.fuscus have evolved to adapt to their specific hypoxic environments.Of which,L.mandarinus is mainly in oxidative stress,metabolism,and DNA damage repair.Except for these three similar aspects,N.fuscus also includes purine metabolism,apoptosis and redox.This may be due to the relatively simple underground tunnel environment with low-oxygen but relatively stable temperature and humidity.However,the environmental factors such as low temperature and strong ultraviolet light associated with low oxygen in the plateau are obviously harsh and more complicated.Our results show that both subterranean rodents and plateau mouse have adapted to the hypoxic environment in terms of oxidative stress,metabolism,and DNA damage repair.2)L.mandarinus shows stronger oxygen sensation and regulation ability to environmental hypoxia,while L.brandtii shows negative response to hypoxic stress.Acute hypoxia causes L.mandarinus to induce the stress responses and impair the metabolic processes;which makes L.brandtii more damaged,upregulate the immune response,stress response and downregulate DNA repair process.Chronic hypoxia could activate the HIF signaling pathway in L.mandarinus but down-regulate neuropathy-related pathways,while chronic hypoxia treatment makes L.brandtii increase the ability of neurotransmitter's regulation,immunity,signaling transduction and hypoxic stress response.Our results demonstrate that L.mandarinus has a stronger ability of sensing and self-regulation upon hypoxia than L.brandtii,which shows stress response and this effect increases as the environmental hypoxic intensity turns to high.3)N.fuscus have better adaptability to plateau hypoxia by down-regulating oxygen-consuming processes such as immunity,improving the response of cell cycle and DNA damage stimuli to quickly renew cells,and maintaining the body's normal function;L.brandtii upregulates stress responses to hypoxia and cold,glycolysis processes,metabolic pathways and related functions.In conclusion,our research shows that sustained and stable environmental hypoxic pressure can cause N.fuscus and L.mandarinus to produce similar molecular evolutionary mechanisms,while changes in the environmental oxygen content can cause species to produce diverse physiological response mechanisms. |
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