A Transcriptomics Study On The Adaptation Of Phrynocephalus Vlangalii To High Altitude Hypoxia And Low Temperature

The Qinghai-Tibet Plateau(QTP)is the highest plateau in the world,known as “roof of the world”.High-altitude environments present a formidable challenge to their native animals due to its hypoxia,cold temperature and strong ultraviolet(UV)radiation.Living on the plateau for many generations,plateau indigenous animals have evolved their unique adaptation mechanisms from phenotype to genetics,physiology to molecular mechanisms.Phrynocephalus vlangalii is an endemic species of the QTP,which has been well adapted to the plateau environment through long time of evolution.In the present study,RNA-Seq was used to conduct a comparative transcriptomic study on different developmental stages(juveniles and adults)between Maduo(4270m)and Delingha(2862m)populations of P.vlangalii.The differentially expressed genes(DEGs)were selected for further functional GO and KEGG function analysis to screen key genes and metabolic pathways in order to further explore the adaptability of reptiles to high altitude and their regulation mechanism.After assembly and filtration of the raw data,a total of 84.05 Gb Clean data was obtained with each sample reached 6.34 Gb from 12 samples of juvenile and adult from low-and high-altitude of P.vlangalii.The percentage of Q30 was 90.64% or more.96348 Unigenes were obtained after assembly by Trinity software with a N50 of 1852 bp.With functional annotations to Unigenes,including alignment with NR,Swiss-Prot,KEGG,KOG,eggNOG,GO,and Pfam databases,19947 Unigene annotations were obtained.Four groups of samples were compared in pairs.Using FDR<0.01 and FC?2 as criteria,differentially expressed genes(DEGs)were screened among the samples and their functional annotations and metabolic pathways were analyzed.The key metabolic enzymes and substances of energy metabolism were determined by physiological and biochemical methods.The transcriptome and biochemical methods were combined to explore the high elevation adaptation(HEA)of different populations and different developmental stages of P.vlangalii.In juveniles,we obtained 688 DEGs in the comparison of Delingha and Maduo populations,these DEGs are annotated to 60 GO terms,with which the molecular chaperone-dependent protein folding(GO:0051085),oxygen binding(GO: 0019825),oxidative stress response(GO: 0006979),and ATP catabolic process(GO: 0006200)were significant enriched.These DEGs involved a total of 78 KEGG pathways.Enrichment analysis indicated fatty acid metabolism(ko01212)was the enrichment pathway.The genes involved in fatty acid synthesis(ACC2 and SCD)were up-regulated while the genes involved in fatty acid oxidation(CPT1)are significantly down-regulated.Measurement of metabolic enzyme activity and metabolites content showed that the high-altitude population had lower activity of HOAD and higher total cholesterol content.These results indicate that the juveniles of high-altitude P.vlangalii are more inclined to the synthesis and storage of fatty acid.In adults,2223 DEGs were generated between Delingha and Maduo populations,then were annotated into 54 GO terms,of which ERK1 and ERK2 cascade regulation process(GO: 0070372),electron carrier activity(GO: 0009055)and heme binding(GO: 0020037)were significantly enriched.These DEGs involved in 185 KEGG pathways,in which the neuroactive ligand-receptor interaction(ko04080)and phenylalanine,tyrosine and tryptophan biosynthesis(ko00400)were highly enriched.DEGs involved in pathway of fatty acid degradation(ACAT2 and AOX1)and biosynthesis of amino acids(TAT,ARG2 and ASS1)are up-regulated in high-altitude populations.The results of enzyme activity assays show that high-altitude populations have higher LDH and HOAD activity.These results indicate that the high-altitude adult P.vlangalii are more prefer to use fatty acids as a fuel to supply energy,which is consistent with the results of Xiaolong Tang(2013).In Delingha population,1069 DEGs were identified between juveniles and adults,and annotated into 41 GO terms.The immune system process(GO: 0002376),cell growth(GO: 0016049)and complement activation pathway(GO: 0006957 and GO: 0006958)were significantly enriched.These DEGs involved in 215 KEGG pathways,in which the mineral absorption(ko04978),phagosome(ko04145)and oxidative phosphorylation(ko00190)pathways were highly enriched.Among them,the expression levels of fatty acid biosynthesis related genes(ACC1,FAD6 and SCD)in adults were up-regulated,and the expression levels of fatty acid oxidation-related genes(CPTIB and ACAT1)were down-regulated.The result indicated that the adults tend to enhance the synthesis and storage of lipids.Adults also up-regulated most of the genes involved in the immune system process.The immune system enhancement can effectively prevent the lizards from the invasion of virus and bacteria and make them better adapt to the complex environment of habitat.In Maduo population,there are 3019 DEGs between adults and juveniles being annotated to 48 GO terms.Among them,glycolysis process(GO: 0006096),immune system process(GO: 0002376)and growth factor activity(GO: 0008083)were enriched.The DEGs were involve into 184 KEGG pathways,in which oxidative phosphorylation(ko00190),steroid biosynthesis(ko00100),and cardiac muscle contraction(ko04260)were significantly enriched.Compared with juveniles,the expression levels of fatty acid synthesis genes(FAD,ACC2,ACSL3 and ELOVL)in the adult were down-regulated,and fatty acid oxidation genes related genes(KCS and AOX1)were up-regulated.In addition,the adult also down-regulated the expression levels of genes related to the oxidative phosphorylation(ND,NDUFS,SDHUFS2 and COX)and glycolysis(LDHA,LDHB,GAPDH).These results indicate the adult P.vlangalii tend to enhance the oxidation of fatty acid for energy supply.We also compared the transcriptomes of the livers of P.vlangalii in three periods of hibernation(pre-hibernation,hibernation and arousal from hibernation),screened DEGs and clustered them to find out the key metabolic pathways involved in hibernation regulation.After the filtration of the transcriptome raw data,a total of 41.41 Gb clean data was obtained.These reads were then assembled,generating 192696 Unigenes.Through annotation,a total of 26566 Unigenes annotated based on the databases mentioned above.The pairwise comparisons of 3 groups of samples were made.DEGs were screened by FDR<0.01 and FC?2,and functional analysis of these DEGs was performed.The results showed that the genes related to fatty acids metabolism(ko01212)were significantly up-regulated,while the genes related to the oxidative phosphorylation(ko00190)and glycolysis process(ko00010)were down-regulated in the hibernation group compared with the other two groups.It suggests that energy was provided mainly by fatty acid oxidation during hibernation,which is consistent with the results of Yanhong Xiao(2016).In addition,during hibernation,the expression of immune-related molecules(IL8,CXCL1 and FHC)is up-regulated to protect the body from bacterial infection.These results indicated that hibernating P.vlangalii can tolerate and adapt to the extreme environment such as low temperature during hibernation through regulation of metabolism,immunity response and antioxidant system.The present study clarifies the gene expression of the adaptation to plateau environment and the low temperature during hibernation in P.vlangalii.The key genes and metabolic regulation pathways related to metabolism,immunity and growth regulation were determined by screening the perspective of transcriptomics.On the one hand,our present study can provide a reference and basis for the further verification of physiological and biochemical perspectives;on the other hand,it can enrich the transcriptome data resources of different developmental stages and different physiological states of the P.vlangalii,providing utilization information for large-scale data analysis.