| Oxygen is the essential element for most organisms on earth.Once the oxygen supply was insufficient,tissues or cells could suffer from hypoxia,followed by a stress response of the body.Hypoxia could affect the physiological/pathological process of animals,as well as growth and development process.Underground tunnels have natural hypoxia environment for subterranean rodents to live in,which made subterranean rodents evolved adaptive evolution in their morphology,physiology,behavior and genetics.Lasiopodomys mandarinus is a typical subterranean rodent,which lived underground for its entire life.However,its congeneric species L.brandtii have much aboveground activities.This makes these two species ideal contrast animals in the study of hypoxic adaption.This research was taken L.mandarinus as objects and L.brandtii as contrast.In this thesis,status and environmental parameters of L.mandarinus tunnels were measured to verify its natural living condition.In addition,phylogenetic analyses based on mitochondrial genome were executed to verify the relationship between L.mandarinus and L.brandtii in molecular evolution level.After that,the brain transcriptome of the L.mandarinus and L.brandtii under normoxia,chronic hypoxia and acute hypoxia environment were sequenced.Comparative transcriptome analyses were used to investigate different adaptive strategies for L.mandarinus and L.brandtii under chronic hypoxia and acute hypoxia.Moreover,the complete genome of L.mandarinus was sequenced and used as reference genome to do the transcriptome analysis.Correlations between two gene expression results with or without a reference were compared to confirm the consistency between these two methods.At last,transcriptome data of L.mandarinus and L.brandtii,the genome data of L.mandarinus were collected to build a database.The database have functions like storage,management,retrieval and remote access,as well as data sharing and visualization.Main results of this thesis are as follows:1)A typical tunnel system of L.mandarinus was about 40 meters in length,which divided into two main parts: foraging tunnel with depth of 20~30 cm underground,and the nest with depth of 60~80 cm underground.Temperatures inside the tunnel were 24±2?,humidities were over 90%.The oxygen levels inside the tunnel were remained at 17.24%~19.48%,and the minimum oxygen level as 16.04% was detected after rain.Tunnel' carbon dioxide levels were between 0.03%~2.55%,which have negative correlation with tunnel' oxygen level.2)Phylogenetic analysis based on mt DNA showed that L.mandarinus and L.brandtii had a closest relationship in molecular evolution level,these two along with Microtus gregalis and Proedromys liangshanensis formed a clade that separated from genus Microtus.3)High throughput sequencing generated 135 Gbp high quality data from 18 brain samples of two species.Transcripts(unigenes)of L.mandarinus and L.brandtii were de novo assembled based on merge assembly method.N50 of these two transcripts were over 2,200 bp,represented a well assembly efficiency.20,172 and 19,215 unigenes were annotated in one or more databases.The results of Nr annotation showed that 2% of the annotated unigenes of L.mandarinus had closest homology with another subterranean rodent Spalax galili.4)We have identified 695 DEGs(differentially expressed genes)from chronic hypoxia group versus normoxia group in the brain transcriptome of L.mandarinus,which was higher than 158 DEGs in L.brandtii.The results of function annotation,function enrichment,pathway enrichment and gene-pathway network analysis were as follows: after long time exposed to chronic hypoxia environment,genes that control functions like oxygen transportation,angiogenesis,anti-angiogenesis,DNA repair,apoptosis and autophagy were up-regulated in the brain of L.mandarinus.Meanwhile the genes control of neurocyte protection,CO2 damage prevention,blood pressure and neuronal activities highly up-regulated.Along with down-regulation functions of proteolysis and coenzyme metabolism.As for the L.brandtii under chronic hypoxia,several genes control of oxygen transportation,angiogenesis,anti-angiogenesis,neurocyte protection and CO2 damage prevention were up-regulated,but dopamine synthesis and transportation genes were down-regulated.DEGs for L.mandarinus were superior in cellular level,indicated that L.mandarinus has more sensitive and positive hypoxia adaption strategies than L.brandtii.5)Compared to normoxia,1,386 and 1,879 DEGs were identified in the brain transcriptome of L.mandarinus and L.brandtii under acute hypoxia.The results of function and pathway enrichment were similar between these two species.The up-regulated DEGs were mainly function in angiogenesis,anti-angiogenesis,anaerobic respiration,DNA repair,cell proliferation and apoptosis.Differences in L.mandarinus were up-regulation of neurocyte protection and more number of anti-angiogenesis control genes,down-regulation of dopamine synthesis and transportation genes,as well as a set of tumor growth and anti-tumor genes which had opposite expression pattern in tumor microenvironment.As for the L.brandtii,immune response genes were up-regulated but several of neurocyte protection genes were down-regulated,indicated that the response strategy for acute hypoxia in L.mandarinus was more efficient than L.brandtii.6)High quality data with genome coverage of 140 X were obtained from whole genome sequencing of L.mandarinus.After assembling,2.10 Gbp Contigs(N50 = 51.15kbp)and 2.15 Gbp Scaffolds(N50 reached 6.15Mbp)were generated,which represent the length of complete genome.7.77 million SNPs were identified through SNP calling.Annotation based on homology alignment and de novo prediction identified a total of 33.93% repeats,21,229 coding genes and more than 7,000 non-coding RNAs.Comparation between gene expression results in transcriptome analysis with or without a reference showed a significant positive correlation(r = 0.836,p <0.0001).7)The transcripts and annotation data of L.mandarinus and L.brandtii were sorted and stored in an Access database.A local BLAST database that contained two transcripts and genome sequences of L.mandarinus was constructed.These datasets were integrated and visualized by use Java programming language and Apache + Tomcat Server,finally achieved the goal of remote access from local network.Conclusions:1)Tunnel system of L.mandarinus provided a stable temperature and high humidity living environment,along with long-term hypoxia and high CO2 stress.These parameters were similar to tunnel system of species within genus Spalax.2)L.mandarinus and L.brandtii both belong to genus Lasiopodomys,these two along with Microtus gregalis and Proedromys liangshanensis formed a clade that separated from genus Microtus.3)The unigenes of L.mandarinus and L.brandtii were very much alike in every aspect,which may be because of their closest relationships.The results of Nr annotation showed that 2% of the unigenes of L.mandarinus had closest homology with another subterranean rodent Spalax galili,which could be the convergent evolution in hypoxia adaptions.4)L.mandarinus has evolved strong adaptability to acute and chronic hypoxia compared with L.brandtii.The mechanisms of hypoxia adaptions in molecular level were: efficient energy utilization,strict angiogenesis/anti-angiogenesis regulation and various DNA repair and apoptosis regulations in cellular level.5)L.brandtii had also evolved a certain degree of hypoxic adaptations;abilities such as angiogenesis and oxygen transportation were enhanced under hypoxia environment.However,its regulations of energy metabolism and cell proliferation were weaker than L.mandarinus.6)Whole genome sequencing of L.mandarinus generated high qualified assemble and annotation results.Comparation of gene expression results between transcriptome analysis with or without a reference showed a significant consistency.7)The L.mandarinus transcriptome and genome database have realized functions for data visualization and remote access. |
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