Study On Blood Transcriptome And Genome-wide Methylation Of Giant Panda At Different Ages

The giant panda(Ailuropoda melanoleuca)is China's unique national Class ?endangered and rare wild protected animal,as well as the symbol and flagship species of world biodiversity protection.Over the past 20 years,Chinese scholars have conducted a number of researches on blood physiological and biochemical indicators of giant pandas.However,the regulation mechanisms underlying age-related changes in the blood physiological and biochemical indicators have not been reported.Therefore,in this study,the blood of giant pandas at young,adult,and old ages were taken as the research object to explore the changes in the blood physiological and biochemical indicators at different ages,and RNA-Seq and WGBS technologies were used to construct the blood transcriptome and methylation maps at three ages,and to screen the differentially expressed and methylated genes among young,adult and old giant pandas and their functions.The main findings are listed as follows:1.The analysis of blood physiological and biochemical indexes of giant pandas at different ages(young,adult and old)showed that the number of white blood cells,neutrophils and hemoglobin rise firstly and then decrease with age,indicating that the immune function of giant pandas are gradually improved from young to adult,but gradually decreases from adult to old.Furthermore,both serum sodium and chloride decrease with age,while aspartate aminotransferase and glutamyl transpeptidase increase significantly from adult to old(P<0.05),indicating that inflammation gradually increases and accumulates with age;glucose metabolism level gradually decreases with age,and compared with the young giant pandas,the blood glucose metabolism of the old ones decreased significantly.2.RNA-Seq was used to construct the transcriptome atlas of blood in the giant pandas at three different ages.There were 716,373,and 1133 differentially expressed genes identified between young vs.adult,adult vs.old a,and young vs.old age respectively.Different expression levelsof the genes belonging to the CXC,interleukin,and tumor necrosis factor gene family betweeen young vs.adult imply their key role in influencing changes in immune function during growth and development of giant pandas.Changes in the immune function of giant panda between adult vs.old may be related to the cascade pathway of complement and coagulation system.Based on the changing trend of differential gene expression throughout the three ages,it was found that the chemokine CXCL12 may be an important gene that mediateage-related changes in the immune function of giant panda.3.A single-base resolution DNA methylation expression profile of the whole panda blood genome was constructed by WGBS technology,and its DNA methylation characteristics were mainly manifested in: CG was the main type of panda blood methylation,and very few occurred on CHG and CHH sequences;the distribution pattern of DNA methylation levels at different ages was similar: under the CG sequence environment,the DNA methylation levels in introns and 3'UTR were higher,followed by CGIshore,repeat and in the exon region,the DNA methylation level of the promoter region was the lowest.4.By analyzing changes in the methylation of giant panda blood at different ages,it was found that under the CG sequence environment,the DNA methylation level of the giant panda decreased with the age.The young vs.adult,adult vs.old and young vs.old groups identified 4273,6544 and 8834 differential methylation regions(DMRs),respectively,corresponding to 448,750 and 851 differential methylation genes(DMGs),respectively.Functional enrichment analysis showed that DMGs between young vs.adult were mainly enriched in metabolic and immune-related pathways,and DMGs identified between adults vs.old were mainly enriched in disease-related pathways.DMGs between young vs.old may affect the immune and metabolic functions of giant pandas mainly through the C-type lectin receptor pathway and the cortisol synthesis and secretion pathway.5.Combined analysis of methylation and transcriptome data showed that 19,17,41 genes with differential expression and differential methylation were found between young vs.adult,adult vs.old,young vs.old,respectively.Function analysis showed that these genes were related to immunity and metabolism,indicating that DNA methylation may affect the immune and metabolic functions of giant pandas at different ages by regulating the expression of these genes.Further exploration of the gene with a differentially methylated promoter and its regulatory relationship with gene expression showed that during the growth and development of giant pandas,changed methylation levels in Cp G island region of the promoter of the RAPGEF3 gene may inhibit the expression of RAPGEF3 gene itself and indirectly regulate the expression of CXC gene family genes.In summary,results from this study indicated that DNA methylation is responsible for age-related changes in the immune and metabolic functions of giant pandas by regulating gene expression.Among them,the methylation status of the RAPGEF3 gene may play a more important role from young to old.These results provide a theoretical reference for the research of epigenetics and the protection of giant pandas.