Cloning Of EPO Gene From Myospalax Cansus And Expression Of Its MRNA In Brain Tissue

Myospalax cansus is a unique subterranean rodents which lives on the loess plateau of our country,The living environment is typically low O2and high CO2all their life,Living long term in the hypoxia underground environment,they have developed a series of unique physiological mechanisms to adapt to the underground low oxygen environment, is an ideal animal model to study mechanism of hypoxia adaptation.Research shows that M. cansus through the proliferation of red blood cells and synthesis of a large number of hemoglobin to improve the ability of carrying oxygen of blood,reduce the hypoxic damage to itself,The formation of red blood cells mainly regulate by erythropoietin (erythropoietin, EPO),EPO by stimulating bone hematopoietic function,to increase the number of red blood cells so as to improve the ability of blood to carry oxygen and increase the oxygen utilization in the body,improve the tolerance of animal to hypoxia,The low oxygen is one of the important factors for promoting the EPO generate,Shams (2004) et al do researche on mole rat (Spalax) about erythropoietin and hypoxia-inducible factor-la hormone, showing that subterranean rodents can well adapt to the underground hypoxic life environment at the genetic level.In recent years, the study found that EPO and its receptor expressed in the central nervous system, has many nerve protective effect.many function of neurons is regulated by the EPO and EPO has been considered to be a new signal transduction molecule in the central nervous system.In this paper, the mRNA expression levels of EPO under hypoxia in the brain tissue were investigated by real-time RT-PCR;the full-length cDNA of EPO gene is coloned from the Myospalax cansus by RT-PCR;The bioinformatics method is used to analyse characteristics of full length cDNA and predict functional motifs in the ORF. The major experiment results are as follow:1. Using the RQ-PCR technology research the expression of EPO mRNA under different hypoxia group in M.cansus and R.non>egicus brain tissue. The results show that the M.cansus brain tissue normoxia group express the minimum amount of EPO mRNA,the acute hypoxia group expression increased significantly, the chronic hypoxia group expression decreased than acute hypoxia group, but still higher than normoxia group; the R.norvegicus brain tissue normoxia group express the minimum amount of EPO mRNA,the acute hypoxia group expression increased extremely significantly, the chronic hypoxia group expression increased extremely significantly than acute hypoxia group. M.cansus EPO expression is less affected by hypoxia, M.cansus has good tolerance to hypoxia, is not sensitive to hypoxic stimulus, declare that it can maintain the normal physiological function under hypoxia environment by rapid feedback mechanism.Thus, we deduced that the M.cansus more effectively mobilize the body’s feedback mechanism than R.norvegicus to adapt to hypoxic environment. Because the EPO has many nerve protective effect in the central nervous system, M.cansus more adapt to the hypixia than SD rats and then reduce the hypoxic damage to the brain tissue, this study laid a certain foundation about the molecular mechanisms of EPO in the brain tissue. The different expression between them, probably because the different molecular structure of EPO.2. The full-length cDNA of M.cansus EPO sequence is579bP,The EPO gene cDNA sequence homology is high up to99%with the published Eospalax baileyi,So sure the cDNA sequence gained is the M.cansus EPO gene. Through ORF Finder online tool to predict ORF,including an open reading frame(ORF)of579bP, which deduced encodes a192amino acid peptide. So we determine the gained sequence is the full-length cDNA sequence of M.cansus EPO gene,Can encode a complete EPO protein sequences and can undertake follow-up experiments and data analysis.3. Bioinformatics analysis the EPO proteins sequence. The physicochemical property analysis shows the molecular weight of EPO protein is21357.8Da and theoretical isoelectric point (PI) is7.64respectively;its chemical formula is C960H1543N257O276S8.Signal peptide analysis showed that the protein with a signal peptide site, shows that the protein type belongs to secrete proteins. The functional group of the protein sequence analysis, found that the protein sequence with multiple function sites, Mainly includes three N-glycosylation sites, two casein kinase Ⅱ phosphorylation sites, one cell attachment sequence site and erythropoietin/thrombopoeitin signature site; The secondary structure of EPO protein was determiened by bioinformatics software, and the resμlt displays that it is a kind of all-alpha protein. Three-dimensional structure analysis show that the protein sequence with the best match template is1buyA, the best talignment region locate in the27-192amino acid residues, sequence consistency is80.12%, evalue is4.54e-70. The space conformation of EPO is composed of four alpha helix beam and three cyclic peptide chain.ClustalX homology comparison results show that the amino acid sequence of EPO in M.cansus,E.baileyi,Israel blind mole and the underground rats have the highest homology, but lower homology with hamster, Microtus oeconomus and people; Although M. cansus, M.baileyi belongs to the genus Myospalax of the family Cricetidae and Spalax allospecies to the family Spalacidae, the phylogenetic tree of EPO showed that these two subterranean species converged into one branch, genetic distance and molecular phylogenetic tree showed that M.cansus、E.baileyi and Nannospalax ehrenbergi have the minimuM genetic distance and has the closest evolutionary relationship, the farthest volutionary relationship to human.EPO gene function differentiation and function of site analysis of underground and ground rat showed there EPO gene functional differentiation between them, with a total of29amino acids of the function of the a posteriori probability value more than0.80differentiation sites, there are two (117leu,161val) the function of the a posteriori probability value more than0.90differentiation decided to site. M.cansus gene selection pressure analysis showed that EPO gene in evolution mainly by purifying selection and neutral choice, in this kind of highly conserved in evolution.Site model to detect the three are choosing amino acid sites, respectively for the11th leucine,144th of alanine and152-bit proline(11Leu,144Ala,152Pro).From underground and ground rat EPO gene function and selection pressure analysis can be seen, underground and ground rat in the long process of evolution, some amino acids sites have function differentiation and positive selection adaption. These amino acid sites in EPO gene is convergent evolution driven by similar hypoxic ecological niches, may be one of the molecular mechanisms to adapt to harsh underground hypoxia environment, provides reference for study the EPO gene function of the hypoxia adaptation mammals, easy to further study the relationship between structure and function as well as the hypoxia adaptation and resistance, Provide the theory basis to clinical medicine, aviation medicine, sports medicine and basic research and treatment for diseases caused by hypoxia.