| Myospalax cansus is subterranean rodents which lives on the Loess Plateau of our country. Belong to the genus Myospalax family of Rodentia, Spalacidae, Myospalacinae, Myospalax. And it survives in underground burrow all life long. Living long term in the hypoxia underground low O2 and high CO2 environment, we know that they adapt underground lives in both physiological and biochemica levels. So Myospalax cansus is good materials for hypoxia research. Methionine sulfoxide reductase (MsrA) is a kind of small within the cytoplasm of enzymes which is widespread in organisms. MsrA is an important part of antioxidant system in the body, and one of its main function is to repair proteins and remove ROS, making a functional protein from oxidative damage by reactive oxygen species, so as to maintain its normal biological activity. Studies have shown that MsrA has strengthen the role of resistance to oxidative in cells or species. MsrA gene has highly homologous between different species, and different species of MsrA amino acid sequence is very conservative. MsrA exists in all organizations in mammals, but expresses in different organizations.In this paper, using the real-time RT-PCR technique to investigate MsrA mRNA expression levels under hypoxia in brain and liver tissue of Myospalax cansus and SD rats; The full-length cDNA of Myospalax cansus MsrA gene was coloned by RT-PCR technique, and the amino acid sequence was predicted; Using bioinformatics method, Myospalax cansus MsrA protein sequences was analyzed. The major experiment results are as follow:1. Using the real-time RT-PCR technique to research MsrA mRNA expression levels under hypoxia in brain and liver tissue of Myospalax cansus and SD rats. The results show that the MsrA mRNA expression of acute hypoxia group increased significantly relative to normoxia group both in Myospalax cansus and SD rat liver tissue. Chronic hypoxia group had no significant difference relative to normoxia group, but it had significantly decreased relative to acute hypoxia group. That acute hypoxia can stimulate the liver tissue MsrA mRNA expression, Myospalax cansus and SD rats each organ in the body and the regulation system of the body to adapt changes in different levels of integration. Compared with Myospalax cansus, acute hypoxia of SD rats increased more significantly. It instructed that acute hypoxic stimulus response of SD rats was more intense, it may be that the Myospalax cansus adapt to underground low oxygen environment for a long time, so to relative SD rats, the liver oxygen pressure drop on the outside stimulation was not sensitive with good tolerance to hypoxia. However, the MsrA mRNA expression of acute hypoxia group had no significant difference relative to normoxia group in Myospalax cansus brain tissue. Chronic hypoxia group expression increased significantly relative to normoxia group and acute hypoxia group in Myospalax cansus brain tissue. This instructed that acute hypoxia treatment cannot stimulate the MsrA mRNA expression in brain tissue. The body may be through other physiological responses to adapt to the severe hypoxia environment. Raising MsrA mRNA expression level in brain was usually the sign of adapting to chronic hypoxia environment. Speculated that this was one the mechanism to anti-aging and keeping longevity for Myospalax cansus. MsrA mRNA expression in SD rats had, three groups had no significant difference between treatment group. Speculated that SD rat cell anti-oxidation ability is limited, and acute hypoxia had cause serious damage to the body, so the body cannot effectively to repair the damage. In addition, the MsrA mRNA expression in brain was higher than liver in three groups. This apparent tissue specificity, and MsrA may had special physiological functions in the brain tissue.2. The full-length MsrA gene cDNA sequence of Myospalax cansus was 699 bp, containing an open reading frame, which deduced encodes 232 amino acids. The MsrA gene cDNA sequence homology was high up to 91% with spalax using BLAST. It was consistent with ORF finder on the sequence processing online toolkit (SMS). So we can determine preliminary that the gained sequence was the full-length cDNA sequence of Myospalax cansus MsrA gene and it can encode a complete MsrA protein sequences and undertake subsequent experiments and data analysis.3. Bioinformatics analysis the MsrA protein sequences. The physicochemical property of MsrA protein was analyzed by the ProtParam. The results showed its chemical formula was C1145H1764N328O326S10; the molecular weight of MsrA protein was 25.7kDa, and theoretical isoelectric point (PI) was 9.33; it had 3573 atoms; MsrA protein was stabilize the protein by Guruprasad method. Analysing the MsrA protein signal peptide by SignalP 3.0 server software analysis, the result showed that the MsrA protein had no signal peptide, so the protein was not secreted protein. Use TMpred to analyze the across membrane area of Myospalax cansus MsrA, the result showed that the protein across the membrane area. Predicting the subcellular localization of proteins, found that the protein secretion pathway was M type, namely, positioning in the mitochondria. Using the Hphob./Kyte & Doolittle method through ProtScale to calculate hydrophobic, found that the whole protein hydrophobic maximum value was 1.900, and the minimum value was 2.500. The protein coiled coil analysis through the COILS Server forecast, found that the Myospalax cansus MsrA proteins had a typical coiled coil structure. Using the CDD to predict Myospalax cansus MsrA protein conservative domain structure, the results showed that it had phosphoribulokinase (PRK) conservative domain, and it belong to the Peptide Methionine Sulfoxide Reductase (PMSR) superfamily. The functional group of the protein sequence was analyzed through PredictProtein, found that MsrA protein sequence had multiple function sites, mainly included two protein kinase C phosphorylation sites、two casein kinase Ⅱ phosphorylation sites、seven N-glycosylation sites. The secondary structure of MsrA protein was a kind of mixed protein, composed of alpha helix and irregular curl. Tertiary structure was determiened by SwissModel automatic mode (Automated Mode), the result showed that the best match template of MsrA protein sequence was 1fvaB, and the best talignment region locate in the 28-227 amino acid residues, sequence consistency was 83.58%, evalue was 2.07e-94.ClustalX homology comparison in Myospalax cansus, Heterocephalus glaber, Homo sapiens, Ochotona princeps, Saimiri boliviensis, Jaculus jaculus, Microtus ochrogaster, Mus musculus, Rattus norvegicus, spalax galili, results found that Myospalax cansu MsrA gene had the highest homology with spalax galili,91%. And its homology with Jaculus jaculus and Microtus ochrogaster were 86%, with Homo sapiens、Saimiri boliviensis and Mus musculus were 85%, Heterocephalus glaber、 Ochotona princeps and Rattus norvegicus were 84%. This suggested that MsrA gene was conservative and the evolution of the sequence was similar between various animals. ClustalX homology comparison in the sequence of MsrA amino acids sequence of the species, found that Myospalax cansus also had the highest homology with spalax galili,92%. And its homology with Ochotona princeps, Jaculus jaculus, Saimiri boliviensis, Microtus ochrogaster, Homo sapiens, Heterocephalus glaber, Rattus norvegicus and Mus musculus were 88%,85%,83%,83%,81%,81%,81% and 80% respectively. MsrA phylogenetic tree showed that Myospalax cansus and spalax galili had the minimum genetic distance and the closest evolutionary relationship. The genetic distance of Myospalax cansus was far away from the other species, and the furthest genetic distance with Rattus norvegicus.Using DIVERG 3.0 software to calculate the MsrA gene functional differentiation coefficient of underground and ground rat.0=0.8144, this instructed that MsrA existed obvious functional differentiation between them. Theoretically predicted the some key points, the results showed that a total of 48 functional differentiation site. There were 48 amino acids of the function more than 0.9 differentiation site, and 4 amino acids of the function more than 0.95 differentiation site. Myospalax cansus gene selection pressure analysis with PAML software showed that a total of three are choosing sites: respectively for thel5th cysteine,16th of arginine,163th aspartic acid. In the long process of evolution, MsrA gene of Myospalax cansus had the functional differentiation and choosing to adapt to. And this was common ecological niche of convergent evolution with the low oxygen and high carbon dioxide underground. This was adaption to one of the molecular mechanism of underground trunk hypoxic environment. |
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