Analysis Of The Relationship Between Longevity And Telomere Maintenance In Mauremys Reevesii Based On RNA-seq And Telomere Length Measurement Data

Longevity is an interesting object for researchers.As a result of human fascination with their own aging process,senescence has been well studied and documented for many taxa.Many of these examples demonstrate the apparent ability to escape functional impairment,especially reproductive ability,and no increased in age-related mortality.These species were considered as long-lived organisms.Recently,many molecular mechanisms underlying longevity were reported due to the rapid development of high throughput sequencing technique and continuous study on the phenomenon.One of the most important aspect is longevity tightly associated with telomere length maintenance.Many studies suggested that,telomere length undergoes low decrease rate or no significant attrition,and even increased in long lived species.Turtles,as an old clade of taxa,have been widely reported about their long lifespan.Some reliable records announced many organisms had been lived over 100 years.While rare studies reported the mechanisms related to longevity in turtles.One important reason is the lack of large scale molecule data such as genome and transcriptome.In this study,total RNA was extracted from liver,muscle and spleen of three adult(more than 10 years old)Chinese three-keeled pond turtle(Mauremys reevesii)individuals using Trizol reagent.Pooled cDNA library was subsequently constructed and sequenced on Illimina Hiseq 2500 platform.In addition,telomere length in different somatic tissues were compared between sex-matured adults and new born hatchlings(1 year old).A total of 160,998,396 paired-end raw reads of 100 bp length were generated and next assembled into 230,085 unigenes(unique gene).18,846 unigenes yield to known protein coding genes by functional annotation.Candidate genes related to longevity were searched from the total pool of annotated genes.Specially,a series of telomere and telomerase associated genes were screened out from the data,including: telomerase reverse transcriptase(tert),telomerase protein component 1(tep1)and all six shelterin proteins(Telomeric Repeat Binding Factor 1,trf1;Telomeric Repeat Binding Factor 2,trf2;Tripeptidyl peptidase 1,tpp1;Ras-related protein 1,rap1;Protection of telomeres 1,pot1;TRF1-interacting nuclear factor 2,tin2).As catalytic subunit of telomerase,expressed telomerase reverse transcriptase genes may indicated activated telomerase in M.reevesii somatic tissues.Protein encoded by these genes could maintain the stability of telomere and prevent it from degeneration.So they have a vital contribution to the turtle longevity.We analyzed relative telomere length in heart,liver,muscle and intestine tissue of both hatchling and adult M.reevesii individuals using qPCR method.Results showed that no significant changes of telomeres length were found in heart(p=0.454),liver(p=0.230)and muscle(p=0.322)tissue of adults compared with hatchlings.The results could also be a supplement of RNA-seq data.However,telomere length was significantly shortened in adult intestine tissue compared with hatchlings'(p=0.322).And this result might be caused by high level of oxidative stress.As mentioned above,ROS could damage telomere ends and the damage is hard to repair.So further studies are needed to explore the tradeoff between ROS production and telomere maintenance in turtle intestine.In this study,a pooled transcriptome data of M.reevesii was generated,and a series of telomere and telomerase function associated genes were screened out.Telomere lengths in varies of somatic tissues were compared in adult and new born turtles.According to the analysis of RNA-seq data and telomere lengths measurements,we inferred that longevity of M.reevesii might cause by telomerase activity and telomere length maintenance.