Effects Of Mitochondria ATPase Activity And The Expressions Ofβ,γ,δ, Subunits During Hypoxia Stress In Hypophthalmichthys Molitrix

Most of the energy required for the life activities provided by the ATP, and ATP catalysis enzyme plays a key role in ATP synthesis. Mitochondrial ATP is a key enzyme for energy in the life activity of the body, which was composed by two parts of F0and F1. There is an activity to compound ATP with the junction state of F0and F1, while it is decomposed under the unbound state.Meanwhile, the decomposition of ATP drives the rotation of F1, and the flow of protons across the membrane can promote the rotation of F0. Thus the energy of the body which needs to maintain the normal life activities is supplied by the ordered coordination of F0and F1.The increasing activities of agricultural and industrial have caused rising water deterioration including the decreasing of carrying oxygen capacity. The yield of the aquaculture declined consequently, making the oxidative stress reaction of the aquatic animals. Hypophthalmichthys molitrix (silver carp) is one of major freshwater species, which belongs to Cypriniformes, Cyprinidae, accounts for an important proportion in Chinese aquaculture. However, silver carp likes jumping, having lively temperament, hypoxia-intolerance, easily affected by the environmental stress, and prone to occur the phenomenon of floating the head on pond, and so it is a problem to solve nurturing new carp varieties that resistant to hypoxia at this stage. Under hypoxic conditions, energy metabolism is blocked due to the lack of oxygen, fish swimming slowly, so the fish can not meet normal physiological needs. The impact of hypoxia on production efficiency had become a serious problem in high-density aquaculture systems.This paper cloned the β,γ,δ subunits by RACE PCR, analyzed the characterization of these genes expressions by real-time PCR (SYBR green I), and the changes of ATPase activity during hypoxia Stress by oligomycin-inhibitor method, in order to provide a theoretical data to further clarify the response mechanisms of mitochondrial ATP enzyme and the subunits of F1in fish during hypoxia Stress. Results of this study are as follows:1. Effects of the activity of the mitochondrial ATP enzyme under hypoxic stressATPase activities were firstly increased and then decreased in all these tissues (heart, brain, liver, spleen and muscle) during acute hypoxia stress, but the changes are different. The activity of mitochondrial ATPase reached the highest and significantly higher than the control group in brain When DO was3.37mg/L. The activity reached a peak significantly higher than the control group, in heart and muscle as DO being2.11mg/L. While the phenomention of floating head appears (DO:1.12mg/L), the activity reached peak in liver and spleen and significantly higher the control group.2. Cloning and expression of (3cDNA.The β cDNA is1768bp in length with an open reading frame (ORF) of1557bp encoding518amino acid (aa) residues. Multiple polypeptide sequence alignment showed the highest identity with carp (99%), and Danio rerio, Oryzias latipes, Oreochromis niloticus, Takifugu rubripes, respectively,98%,98%,97%,97%. With DO decreasing, the expression of F1-β mRNA was increased first then decreased in heart, brain, liver, spleen and muscle tissues of silver carp. When DO reduced to2.11mg/L, the expression in heart and brain were significantly increased (P<0.05), then having a sharp decline. However the expression significantly increased (P<0.05) in liver, spleen and muscle when DO was3.37mg/L following a sharp drop (P<0.05).3. Cloning and Expression Analysis of y cDNA.The F1-γ cDNA sequence is1154bp long and the ORF is879bp and encodes a protein with292amino acid residues. The amino acid sequence have the highest homology with Danio rerio (93%), followed with Cyprinus carpio, Ictalurus furcatus, Ictalurus punctatus, Salmo salar, respectively, being92%,92%,91%,86%. With DO decreasing, the expressions characteristic of y mRNA showed increased at first and then increased in brain, liver and muscle, the expression was significantly higher (P<0.05) than the control group in the brain and liver when DO was2.11mg/L, significantly higher (P<0.05) in muscle when DO was3.37mg/L. The expression gradually decreased in heart, and significantly lower than the control group (P<0.05), while are showing increasing tendency in spleen, and displayed higher (P<0.05) when DO<2.11mg/L.4. Cloning and Expression Analysis of8cDNA.The position change of8can cause the structure change of F1-ATPase, because δ subunit was the part of the stem-like structure connection F1-ATPase and F0-ATPase. To investigate possible roles of F1-δ subunit in silver carp, the full-length cDNA of this gene was cloned by RACE-PCR, showed that the full-length cDNA of mitochondria ATPase F1-δ mRNA was762bp, containing an ORF of480bp encoding159amino acids. The deduced amino acid sequence exhibited high similarity to Dario rerio (91%), moderate similarity to Salmo salar (84%), Oreochromis niloticus (85%), Oncorhynchus masou formosanus (84%) and Takifugu rubripes (84%). The expressions were gradually downward and significantly lower than the control group in heart (P<0.05), and compared with control group, they were significantly higher (P<0.05) then sharply declined (P<0.05) in brain, liver, spleen and muscle, with DO decreasing. The expression was significantly higher (P<0.05) when expression was up to the highest in liver and spleen.5. Relationship between ATPase activity and β,γ,δ subunitsFrom above experimental results, β,γ,δ subunits of mitochondrial F1ATP enzyme complexes are highly conserved, the changes of the expression of β catalysis subunit have consistent with the varies of ATP enzyme activity in tissues with DO decreasing, that is to say, the expression change of β subunit has the same change of ATPase activity. Expressions of γ,δ subunits was effected by DO reduce, and have differences with the change of ATPase activity in some organizations.