Molecular Cloning And Sequence Analysis Of PGC-1α CDNA In Tibetan Antelope And Tibetan Sheep

Background and ObjectivesThe Qinghai-Tibetan Plateau, is the largest and highest area in this planet, with2.5million km2in size. It includes almost all the world’s territory higher than4500meters. As inclement environment of the Tibetan Plateau, the zoology and botany areconstantly challenged by hypobaric hypoxia, cold temperature, and high UV radiation.However, it’s a paradise to wild animals such as Tibetan antelopes, wild yaks andwild Tibetan donkeys. During their long evolutionary history, those animals havedeveloped their own mechanisms to adapt at the harsh environment.Endemic to the Tibetan Plateau, the Tibetan antelope (Pantholops hodgsonii), belongsto Bovidae family, inhabits open alpine and cold steppe environments between3500and5500meters elevation. They prefer flat, open, terrain, with sparse vegetationcover. They are found almost entirely in China, where they inhabit Tibet autonomousregion, Xinjiang autonomous region and Qinghai province. Because of their peculiarhabitat and habits no zoos or other places in the world have ever tried breading.Tibetan antelopes are good runners and can move as fast as80km/h, with maximumspeeds reported to be110~120km/h. Above all, Tibetan antelope is a perfect subjectfor high altitude adaptation study.As a transcriptional coactivator, PGC-1α can bind to and coactivate most nuclearreceptors as well as many transcriptional factors. PGC-1α is a dynamic regulationfactor through various signal transduction involved in cellular energy metabolismprocesses, including mitochondrial biogenesis and oxidation, hepatic gluconeogenesis,β-oxidation of fatty acids, adaptive thermogenesis, etc. Cold temperature, fasting,endurance exercise could induce PGC-1α expression in specific tissues. However, Itis unclear that what the role does PGC-1α play in high altitude adaptation of theQinghai-Tibetan Plateau native animals. In the present study, we aimed to clone the PGC-1α gene coding regions from Tibetanantelope and Tibetan Sheep, which may provide fundamental sequences data forfurther investigating high altitude adaptation at regulation of transcriptional level inthe future.MethodsIn accordance with approval from the State Forestry Administration (SFA) and theGuide for the Care and Use of Laboratory Animals by the Ministry of Science andTechnology of the People’s Republic of China,9male Tibetan antelopes (Pantholopshodgsonii) were captured from Kekexili Natural Reservation (altitude4300m) and10male Tibetan sheep were bought from Wild Yaks Valley of Haixi state (altitude4300m). Then all the animals were transported to Gurmud (altitude2800m), wherethe physical study was conducted immediately after the arrival of the animals. Afterthat, The hearts from Tibetan antelope and Tibetan sheep were collected. PGC-1αgene nucleotide sequences of Bos taurus, Capra hircus, Homo sapiens, Mus musculus,Sus scrofa, Oryctolagus cuniculus were obtained from GenBank of National Centerfor Biotechnology Information (NCBI).The primers were designed using PrimerPremier5.0and DNAMAN softwares.Total RNAs were extracted from myocardiumof Tibetan Antelope (Pantholops hodgsonii) and Tibetan Sheep. PGC-1α codingcDNA sequences were cloned with reverse transcription polymerase chain reaction(RT-PCR), and the sequences were confirmed by DNA sequencing.ResultsThe cloning and sequencing results confirmed that the PGC-1α gene codingsequences of both Tibetan Antelope and Tibetan sheep showed above90%identitywith other species. In addition, the cloned sequences contained the RNA/DNAbinding sites, RRM (RNA recognition motif), the domains involved in the interactionwith NRF-1and MEF2C, Arg/Ser rich domain, negative regulatory domain, LXXLLmotif, as well as conserved sequences like TPPTTPP and DHDYCQ, which arepresent in all PGC-1family members. Fourteen variable amino acid sites wereidentified in the functional domains mentioned above. Additionally, analysis ofgeneric phosphorylation sites and kinase specific phosphorylation prediction sites indicated that the329threonine amino acid site could be phosphorylated by PKG,which may be unique to Tibetan Antelope. Secondary structures of PGC-1α proteinfrom Tibetan Antelope and Tibetan Sheep were also predicted in this study.ConclusionIn summary, the PGC-1α gene coding regions from Tibetan antelope and TibetanSheep have been successfully cloned (GenBank accession number:JF449959andJF449960), which may provide fundamental sequences data for further investigatinghigh altitude adaptation related to genetics in the future.