Study On Oxidative Stress Response And Changes Of Liver MiRNA In Rachycentron Canadum Under Hypoxic Environment

At present,hypoxia has become one of the main abiotic factors restricting the stable development of Rachycentron canadum aquaculture due to the influence of global warming,seasonal change,diurnal change and eutrophication of water.However,there is still no report on the strategy and response mechanism of cobia in response to hypoxia stress.In order to improve the social benefit and economic value of sergeant fish breeding,it is urgent to breed new varieties with low oxygen tolerance.In this study,juvenile of cobia were selected as experimental subjects.The control group dissolved oxygen(6.64±0.35)mg/L and the hypoxic treatment group [dissolved oxygen(2.64±0.25)mg/L for 3 h of stress] and reoxygenated groups [dissolved oxygen(6.34±0.15)mg/L for 8 h,24 h and 48 h] different experiments.Oxidative stress and energy utilization related enzyme activity indexes were used to preliminarily investigate the oxidative stress response to hypoxic stress.Meanwhile,the full length of Mn-SOD gene c DNA was obtained by Rapid Amplification Of c DNA Ends(RACE)and bioinformatics analysis was conducted to explore the expression rule Of Mn-SOD gene under hypoxic stress.Then mi RNA omics techniques were used to preliminarily screen mi RNA that were involved in hypoxic stress.The purpose of this study is to provide a scientific reference for the breeding of new hypoxic resistant species by thoroughly studying the mechanism of cobia in response to hypoxic stress from the physiological and biochemical level to the molecular level.Main research contents and results are as follows:1.Response of cobia to oxidative stress and energy utilization under environmental hypoxic stress:After hypoxic stress,the activities of malondialdehyde(MDA),catalase(CAT)and glutathione reductase(GR)in the liver were significantly lower than those in the control group(P<0.05),and the activities of lactate dehydrogenase(LDH)were significantly higher than those in the control group(P<0.05).The activities of MDA and LPO in muscle were significantly lower than those in the control group(P<0.05),and the activities of superoxide dismutase(SOD)and LDH were significantly higher than those in the control group(P<0.05).The contents of muscle glycogen and liver glycogen were significantly lower than those of the control group(P<0.01),indicating that the fish body produced oxidative stress response and showed slight oxidative damage.During reoxygenation at 8 h,24 h and 48 h,the contents of MDA,LPO,SOD,CAT,glutathione peroxidase(GPx)and GR in liver and muscle all increased to different degrees.After 24 h and 48 h reoxygenation,the content of glycogen in liver increased first and then decreased.The content of muscle glycogen was significantly lower than that of control group(P<0.05),indicating that the compensation metabolism of the body exacerbated the more intense oxidative stress in the reoxygenation process.2.Cloning and sequence feature analysis of Mn-SOD gene of cobia: The full length c DNA sequence of cobia Mn-SOD gene was 1209 bp,including 684 bp ORF,42 bp 5’UTR and 483 bp 3’UTR,encoding 227 amino acids.Mn-SOD was expressed in all tissues of the fish,among which the expression level of heart was the highest,followed by muscle,brain and liver,and the expression level of spleen was the lowest.During hypoxic-reoxygenation,the expression of Mn-SOD in the liver was lower than that in the control group.Brain tissue is different from the liver,the expression of Mn-SOD in brain tissue was significantly higher than that in the control group after R24 h(P<0.01),but there was no significant difference after S3 h,R8 h and R48 h.3.Analysis of mi RNA associated with the liver of cobia under hypoxic stress: A total of 184 conservative mi RNA and 121 newly predicted mi RNA were obtained by sequencing the livers of the control group(C group)and the hypoxic group(S group).Then the mi RNA expression in response to hypoxic stress was analyzed and 19 differential mirnas(10 differences up and 9 differences down)were screened.KEGG pathway analysis of differential mi RNA target genes revealed that 105(38.46%)metabolic pathways were involved in the regulation of hypoxemia in fish.Among them,12 were involved in amino acid metabolism,such as histidine metabolism(ko00340)and arginine biosynthesis(ko00220).The pathways involved in energy metabolism include starch and sucrose metabolism(ko00500),glycosaminoglycan degradation(ko00531)and galactose metabolism(ko00052).It indicates that in order to cope with the low oxygen environment,the body maintains physiological activities by regulating some important pathways on the transcriptional level,such as the conversion of aerobic metabolism and anaerobic metabolism,the reduction of energy consumption and the promotion of red blood cell proliferation,so as to jointly maintain the homeostasis of the body.