| Our country unique subterranean rats of Myospalax cansus(Gansu zokor) spends its entire life in underground tunnels in which the O2 tension is often severely reducedx. Nevertheless, after a long period of evolution, a lot of adaptive mechanism about physiological and biochemica have been formed. Gansu zokor represents an extreme model of adaptation to an underground environment with hypoxia and hypercapnia. Research have shown that a series of stress leads to increased reactive oxygen radicals as indicating hypoxia environmental conditions and antioxidant system was an important defense system in organisms as a key role in scavenge free radicals and balance of antioxidant system. Superoxide dismutase (SOD), catalase (CAT) and glutathion Peroxidase (GPx) are the most important antioxidant enzyme system in organisms. The research about rodents antioxidant system in hypoxia adaptation mechanism is still very full.This study, we applied the biochemical method compared Gansu zokor and Sprague-Dawley rats(SD rats) SOD, CAT and GPx activity of liver and brain under normoxia, acute hypoxia and chronic hypoxia conditions, and the organization’s total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content. The corresponding genes of those three antioxidase (SOD, CAT and GPx) were detected by Real-time quantitative PCR. And through RT-PCR cloned GPx1 cDNA sequence of Gansu zokor, according to the Gengank, PDB, Pfam, ExPASy, MART, ConSurf database through bioinformatics analysis of nucleotide sequence and amino acid sequence, such as homology modeling, structure comparison, functional site, phylogenetic tree and selective pressure. To investigate the expression of the antioxidant enzymes when organisms in a hypoxia environment, and the antioxidant defense mechanism of Gansu zokor other than ground rats, which provides theoretical basis for hypoxia adaptation of organisms to study. The main results were as follows:1. SOD, CAT, GPx activity, total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content:Under acute hypoxia condition, in the brain of Gansu zokor, SOD, CAT activity and T-AOC were tend to increase (P>0.05), GPx activity was significantly increased(P<0.05), MDA content was tend to reduce (P>0.05), and SD rats changes of these measurement indexes were not obvious in the brain. In the liver of Gansu zokor, SOD and CAT activity were significantly increased(P<0.05), GPx activity and T-AOC were tend to increase (P>0.05), MDA content was significantly increased(P<0.05), CAT and GPx activity were significantly increased(P<0.05) in SD rats and MDA was reduce (P>0.05), besides that, had no significant change found in liver. Under chronic hypoxia condition, in the brain of Gansu zokor, SOD, CAT and GPx activity were significantly increased(P<0.05), T-AOC and MDA had no significant change(P>0.05), GPx activity was significantly increased(P<0.05) in SD rats and had no others significant found in the brain. The changes of liver had similar fluctuation with the acute hypoxia. These illustrate acute and chronic hypoxia condition stimulate the growth of antioxidant enzyme activity in the brain and liver, stabilize the total antioxidant capacity and suppresses growth of MDA. Compared with normoxia, the more significantly increased in chronic hypoxia group than acute hypoxia group. And the enzyme activity increase and MDA content of Gansu zokor were more significantly than that of SD rats, its antioxidant system fit into the low oxygen environment berrer.Under the three oxygen conditions, in the brain, there was no significant difference between both Gansu zokor and SD rats in SOD activity(P>0.05), but GPx activity of Gansu zokor was very significantly lower than SD rats(P<0.001), CAT activity was not significantly different from SD rats in normoxic group, after acute and chronic hypoxia Gansu zokor were significantly higher than SD rats (P<0.05), T-AOC and MDA had no significant differences in this two(P>0.05). In the liver, normoxic group had no significant difference between both Gansu zokor and SD rats in SOD and CAT activity(P>0.05), after acute and chronic hypoxia SOD activity Gansu zokor were significantly higher (P<0.05) and CAT activity SD rats were significantly higher (P<0.05). GPx activity of Gansu zokor was very significantly lower than SD rats(P<0.001), T-AOC was significantly higher than SD rats(P<0.05), MDA had no significant differences in this two(P>0.05). These illustrate the different antioxidant mode between Gansu zokor and SD rats. In the brain they had the same antioxidant capacity, but in liver Gansu zokor had a higher antioxidant level than SD rats, largely based on higher SOD and CAT activity and GPx involved in brain antioxidant mainly than SD rats that in both brain and liver. Moreover. GPx activity increased significantly in hypoxia groups of Gansu zokor’s brain, but was still below the level to SD rats.2. SOD1, CAT and GPx1 mRNA expression levelsUnder acute hypoxia condition, measured mRNA expression showed no significant change compared normoxic except that of SOD1 of Gansu zokor’s brain and CAT of SD rat’s liver were tend to increase(P<0.05). Under chronic hypoxia condition, in the brain of Gansu zokor, SOD1, CAT and GPxl mRNA expression were significantly increased(P<0.05), SD rats had no significant changes except GPxl mRNA expression was significantly increased(P<0.05). In the liver, SOD1 and CAT mRNA expression were significantly increased(P<0.05) both in Gansu zokor and SD rats, GPxl mRNA expression had no significant change(P>0.05). These illustrate the stimulus of chronic hypoxia can more effective cause antioxidant enzyme transcribed than acute hypoxia. Besides, the antioxidant system of Gansu zokor on mRNA expression showed a strong vitality and flexibility under hypoxia stress. It futher compares the mRNA expression of Gansu zokor with SD rats, and had. discovered that Gansu zokor’s SOD1 was very significantly higher than SD rats(P<0.001). The results show clearly that SOD was the main functional component in antioxidant system of Gansu zokor.3. The GPxl gene clone and bioinformation analysisThe full sequence of the Gansu zokor GPx1 gene was 780 bp, including the initiation codon(ATG) and termination codon (TAA), and the size of ORF was 606 bp in length, encoding a protein of 201 amino acids, selenocysteine codon (TGA) located at the forty-seventh amino acid sequence. Besides, the mRNA sequences had type I selenocysteine insertion element (SECIS). Nucleotide and amino acid sequence alignment results showed that, the Gansu zokor GPx1 shared high similarity with Spalax.The amino acids primary sequence of GPx1 protein contain 22 the negatively charged and 23 positively charged amino acid. The relative molecular mass was 22.3KD, isoelectric point (p1)=7.67, molecular formula was C997H1556N278O286S8Se1, belonging to unstable protein. Helix, Strand and Loop serve as the main component of secondary structure, which was a hybrid (mixed) structure. There was no signal peptide and transmembrane region and no two disulfide bond formation. The amino acids tertiary structure showed that GPxl protein combines four groups of similar subunit to form the protein.Secondary structure analysis of the functional domains and functional sites found that, the 14-128 amino acid residues of GPxl was glutathione peroxidase domain, the 35-50 region was the active site, the 72-79 region was conservative sites, and AhpC-TSA, ENT and GAS2 protein domain were also contained. Bisides, the GPxl sequence to best match with TrxR family protein. This could be due to both of them belong to selenoenzyme, that explains the consistency of protein structure and function.Phylogenetic analysis of the the Gansu zokor GPx1 protein was under purifying selection and highly conserved. But also exists significant function differentiation and positive selections. The four amino functional differentiation decisions points where the posterior rate was more than 0.98(QK>0.98), which were the 2Cys, lOGly,158Ile and 199 Gly. And through the site model test and Bayesian method, we predicted four amino acid sites may be subject to positive selection pressure, which were the 129Glu,147Phe, 199Gly,200Ser. These sites might account for the adaptive evolution with underground life about antioxidant enzyme gene of Gansu zokor. |
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