Article Animal Genetics March 2012 Vol.57 No.9: 9971006 doi: 10.1007/s11434-011-4911-3 SPECIAL TOPICS: cDNA cloning and expression of erythropoietin in the plateau zokor (Myospalax baileyi) from the Qinghai-Tibet Plateau WANG ZhenLong1,2†, CHEN Yan3,4†, YANG Jie5, CHEN WeiJun3*, ZHANG YanMing2* & ZHAO XinQuan2* 1 Department of Bioengineering, Zhengzhou University, Zhengzhou 450001, China; 2 Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining 810001, China; 3 Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, China; 4 Graduate University of the Chinese Academy of Sciences, Beijing 100049, China; 5 Department of Epidemiology and Statistics, School of Public Health, Hebei Medical University, Shijiazhuang 050017, China Received August 15, 2011; accepted September 23, 2011 All organisms respond to variation in their environments and manage environmental stress through metabolic adjustments. The plateau zokor (Myospalax baileyi) is an endemic and keystone subterranean rodent species that inhabits the Qinghai-Tibet Plateau between 2800 and 4200 m above sea level. It is a hypoxic-tolerant mammal with a high ratio of oxygen utilization that enables it to cope with its harsh surroundings. To explore the molecular mechanism of altitude acclimatization of the plateau zokor, we cloned the zokor erythropoietin (Epo) gene and used real-time PCR to compare Epo mRNA levels in zokors inhabiting 16 differ- ent altitudes. The full-length zokor Epo open reading frame was 579 bp that encoded a precursor peptide of 192 amino acids with a signal peptide of 26 residues. The Epo gene of the plateau zokor was 81%–95% homologous to that of human, mouse, rat, root vole and the Golan Heights blind mole rat, with the highest homology (95%) to species of the genus Spalax. Epo mRNA was detected mainly in the zokor kidney and spleen among 8 selected tissues. The level of Epo mRNA increased in the liver and kid- ney with increases in altitude. The increase in the kidney was 5 times that in the liver. Remarkably, expression of Epo mRNA in the kidney of zokors living at the highest altitude (4268 m) was 12-fold higher than that of zokors living at the lowest (2492 m) altitude. These findings provide essential information for understanding the possible role of Epo in adaptation to hypoxia in the plateau zokor. erythropoietin, plateau zokor, cDNA cloning, mRNA expression, altitude Citation: Wang Z L, Chen Y, Yang J, et al. cDNA cloning and expression of erythropoietin in the plateau zokor (Myospalax baileyi) from the Qinghai-Tibet Plateau. Chin Sci Bull, 2012, 57: 9971006, doi: 10.1007/s11434-011-4911-3 All organisms have to deal with environmental stress harsh environmental conditions to ensure optimal growth through metabolic adjustment and ultimately they have to and reproduction [3]. adapt to their environment [1]. The Qinghai-Tibet Plateau The plateau zokor (Myospalax baileyi) is an important has an average altitude of 3000 m, making it the highest subterranean rodent endemic to the Qinghai-Tibet Plateau. plateau on earth [2]. Strong ultraviolet radiation, hypoxia It is tolerant of hypoxia and shows a remarkably high oxy- and cold are typical of plateaus and have a profound effect gen utilization ratio to cope with the plateau environment, on animal survival. Over evolutionary time many animals making it a good model for research into adaptation to hy- have developed unique mechanisms in response to these poxia [4]. In their sealed hypoxic-hypercapnic burrow, Zo- †These authors contributed equally to this work. *Corresponding authors (email: [email protected]; [email protected]; [email protected]) © The Author(s) 2012. This article is published with open access at Springerlink.com csb.scichina.com www.springer.com/scp 998 Wang Z L, et al. Chin Sci Bull March (2012) Vol.57 No.9 kors spend their entire life underground, exposed to fluctu- Epo has been cloned from many mammals, including ating O2 and CO2 levels [5,6]. They are active throughout human, monkey, rat, sheep, dog and pig and it shows a high the year, excavating burrow systems and storing food. As is degree of homology among mammals [18,25–27]. However, common in subterranean rodents, the plateau zokor exhibits there have been few reports concerning the Epo gene from morphological, physiological and behavioral adaptations to species living in high altitude, hypoxic environments. The digging and living underground [7]. For example, the zokor plateau zokor is a hypoxic-tolerant mammal with a high has evolved physiological changes to its respiratory and ratio of oxygen utilization. In this study, the Epo gene of the cardiovascular systems to cope with hypoxia [8–10]. The plateau zokor was cloned, and the predicted amino acid se- zokor is dramatically superior to Rattus species in mi- quences were compared with those of other animals. Further, crovessel density of cardiac muscle and myocardial perfor- using real-time PCR, we investigated expression levels of mance, and harbors a much higher capillary and mitochon- Epo mRNA from 8 tissues and across 16 different altitudes drial density [8]. The zokor has a higher erythrocyte count, to explore the significance of Epo in the adaptation of the an increased lung diffusion capacity, and utilizes a myoglo- plateau zokor to the harsh environment of the Qinghai-Tibet bin that is different in structure to that of Rattus [10]. Plateau. Moreover, the number of red blood cells, the density of he- moglobin in the red blood cells and the myoglobin content in heart and skeletal muscle are much higher than in other 1 Materials and methods high-altitude mammals inhabiting a plateau environment 1.1 Experimental sites and animal sampling [11]. Erythropoietin (Epo) is a hematopoietic cytokine that All procedures involved in the trapping and handling of regulates red blood cell production [12]. Epo has 166 amino animals were approved by the Chinese Zoological Society acid residues and a molecular weight of 18.4 kD; however, and were conducted in accordance with the Chinese Practice the overall molecular weight of Epo is 30.4 kD because of for the Care and Use of Laboratory Animals. Eighty-eight three N-linked carbohydrate chains. Two internal disulphide adult zokors (58 females and 30 males) were captured from bonds between cysteine residues 7–161 and 29–33 are 16 locations in the eastern region of the Qinghai-Tibet Plat- known to be necessary for biological activity [13]. Epo is eau over two field excursions from April to June in 2006 expressed mainly in fetal liver and adult kidney [14,15]. and 2007. We collected animals from sites dominated by The regulation of Epo gene expression occurs mainly at the alpine meadows, alpine shrub and farmland where the main transcriptional level by DNA-dependent mRNA synthesis differences were altitude (ranging from 2492 to 4268 m) [16–18] and is controlled by an oxygen detection system and ambient temperature (ranging from −1.3 to 11.8°C). At that responds to changes in venous rather than arterial par- each location we measured elevation, latitude and longitude tial pressure of oxygen (PO2) [19,20]. Clearance of Epo using a handhold GPS (eXplorist210, Magellan Corp, USA) appears to be possible via three routes: excretion through (Table 1). Live-trapping was conducted between 09:00 and the kidney [21], metabolism by the liver [22] or consump- 16:00 h from April to June. Captured animals were firstly tion by the erythron [23]. Epo is essential for the develop- recorded for body weight (g) and body length (mm), then ment of normal erythropoiesis, and underproduction of Epo anesthetized using chloral hydrate (5%) and killed by cer- results in anemia [16]. Regulation of erythropoiesis and red vical dislocation. Dissections were conducted at the trap site. blood cell mass relies on modulating Epo gene expression Eight tissue samples (brain, heart, lung, liver, spleen, kidney, in response to tissue oxygen tension. Developmental, tis- muscle and white adipose tissue) were immediately frozen sue-specific, and environmental signals all contribute to the in liquid nitrogen. precise regulation of the Epo gene. Research on ontogenetic expression of the Epo gene and of hypoxic stress tolerance 1.2 Preparation of total RNA and cDNA synthesis in the subterranean blind mole rat of the Spalaxeh renbergi superspecies has shown that higher Epo mRNA levels exist Total RNA was extracted from a 100-mg portion of frozen in the Spalax fetal liver and kidney compared with Rattus. tissue using TRIZOL reagent (Invitrogen, Carlsbad, USA), In addition, adult Rattus kidney and liver, and adult Spalax and then diluted in 100 μL RNase-free water and analyzed liver express similar levels of Epo mRNA under normoxia by agarose gel electrophoresis. Sharp bands of 18S and 28S and hypoxia [24]. In normoxia, both Spalax and Rattus kid- rRNA were used as indicators of high quality preparations. neys produce small amounts of Epo. Notably, under hypox- RNA concentration was determined by spectrophotometry ic conditions, Epo expression in the adult Spalax hypoxic using a NanoDrop ND-1000 Spectrophotometer (Nano- kidney, the major site of erythropoietin production in adult Drop Technologies, Rockland, USA). RNA samples were mammals, was remarkably higher than in that of Rattus. treated with RNase-free DNase I (TaKaRa, Dalian, China), Therefore, it can be assumed that Epo is a key factor in then stored at −80°C. coping with underground hypoxic stress and that Epo con- Five micrograms of total RNA were used for first-strand tributes to the adaptive strategy of hypoxia tolerance [25]. cDNA synthesis using a SuperScript™ II RT Kit (Invitrogen) Wang Z L, et al. Chin Sci Bull March (2012) Vol.57 No.9 999 Table 1 Geographic and climate data for 88 samples of Myospalax baileyi from 16 sampling locations across the Qinghai-Tibetan Plateaua) Populations Ecogeographical variables No.