Study On The Organism And Molecular Response Of Hypophthalmichthys Molitrix To Hypoxia Stress

In order to explore the organic damage and molecular response of silver carp(Hypophthalmichthys molitrix)under hypoxia stress,in this study,the gill tissue structure was observed histologically,and antioxidant enzyme activities in liver and serum were determined,transcriptome sequencing was conducted to analyze the signal pathways and differentially expressed genes related to hypoxia stress,and the GLUT1 gene was cloned and its expression characteristics was analyzed.The results preliminarily confirmed the effects of hypoxic stress on the organism and the physiological and biochemical and molecular responses of silver carp.The main results were as follows:(1)Effects of hypoxic stress on tissue structure and antioxidant stress of silver carpTo explore the effects of hypoxia stress on the tissue structure and antioxidant enzyme activity of silver carp,in this study,the changes of gill tissue structure and the activities of catalase(CAT),superoxide dismutase(SOD),glutathione peroxidase(GPX)and the expression characteristics of SODs gene were measured under normoxia(DO= 6.42 ± 0.3)mg/L,hypoxia(DO= 0.76 ± 0.03)mg/L,semiasphyxia(DO= 0.58 ± 0.06)mg/L and asphyxia(0.27 ± 0.06)mg/L,respectively.The results showed that hypoxic stress caused different degrees of damage to the gill.With the decrease of oxygen concentration,the gill lamellas gradually exhibited a severe S-shaped curve,in addition,mitochondria-rich cells and squamous epithelial cells were also damaged to varying degrees.With the decrease of dissolved oxygen concentration,the degree of damage was aggravated.The antioxidant enzyme activity system of silver carp was destroyed under hypoxia stress.With the increase of hypoxia stress,the activities of catalase(CAT)and glutathione peroxidase(GPX)in serum increased,and CAT activity reached the maximum at semi-asphyxia(P<0.05),while GPX activity had no significant change.The activity of superoxide dismutase(SOD)was significantly decreased under hypoxia stress(P<0.05).The activity of SOD and CAT in liver reached the maximum at hypoxia,and then decreased to the lowest at asphyxia(P<0.05).The activity of GPX in liver reached the highest level at semi-asphyxia,but there was no significant difference(P>0.05).The expression of Cu/Zn-SOD m RNA in liver was significantly different from normoxia at stages of hypoxia and asphyxia(P<0.05),and the Cu/Zn-SOD expression in gills of hypoxia stress was significantly lower than normoxia(P<0.05).The expression of Mn-SOD in liver firstly increased and then decreased,and significantly decreased in semiasphyxia(P<0.05).The expression of Mn-SOD in gills decreased significantly and then stabilized.Hypoxic stress produced oxidative stress on the silver carp,but it can be regulated by its own antioxidant system.When the dissolved oxygen is too low,the organism will be damaged,or even die.(2)Transcriptome analysis of gill tissue of silver carp under hypoxia stressIn order to explore the molecular responses of gill tissues to different oxygen concentrations,transcriptome sequencing was performed.The results indicate that a total of 2060 differentially expressed genes were identified,including 587 differentially expressed genes(DEGs)in the hypoxia,725 DEGs in the semi-asphyxia,and 748 DEGs in the asphyxia.Only 224 DEGs were co-expressed differently in all treatment groups.KEGG Pathway differential enrichment analysis showed that DEGs were significantly enriched in cytochrome P450 metabolites pathway,FOXO signaling pathway,HIF-1 signaling pathway and MAPK signaling pathway.Cytochrome P450(CYPs)and hypoxia-inducible factor(HIFs)gene families were widely expressed.Seven different genes were selected for q RT-PCR verification,and the results showed that their up-regulation and down-regulation trends were completely consistent with the m RNA sequencing results,which verified the good accuracy of transcriptome sequencing analysis.(3)Cloning and expression analysis of GLUT1 gene of silver carpBased on the GLUT1 gene fragments obtained from the transcriptome,the fulllength of GLUT1 gene sequence of silver carp was cloned by RACE,and the m RNA expression characteristics of GLUT1 in different oxygen concentrations were analyzed by q RT-PCR.The full-length c DNA of the GLUT1 was 2104 bp,encoding490 amino acids,including 34 strongly acidic amino acids(Asp + Glu)and 37 strongly basic amino acids(Arg + Lys).The predicted molecular weight is 53.92 KD and the theoretical isoelectric point(PI)is 8.47.The amino acid sequence consisted of22 serine(Ser),10 threonine(Thr)and 4 tyrosine kinase phosphorylation sites.The GLUT1 phylogenetic tree constructed by Neighbor-joining(NJ)method showed that the GLUT1 of silver carp clustered into a branch with the most similar grass carp,and then clustered into a branch with carp,zebrafish and steelhead,which had the closest genetic relationship,forming a relatively independent large branch,and then clustered into a branch with other fish and lactation species.The expression of GLUT1 gene in brain,gill,heart,liver,kidney,intestine,muscle and spleen was detected by q RT-PCR.The results showed that the relative expression of GLUT1 from high to low was gill,spleen,brain,heart,intestine,kidney,muscle and liver,respectively.the expression level of in gill was significantly higher than brain(P<0.05).Under different oxygen concentrations,the expression of GLUT1 in liver,brain,heart and gills were also different.In the brain,heart,and liver,the expression of GLUT1 in each hypoxic stress group(hypoxia,semi-asphyxia and asphyxia)was significantly higher than normoxia(P<0.05),while in the gill,the expression of GLUT1 in the hypoxia and semi-asphyxia increased significantly and then decreased significantly at asphyxia(P<0.05).In the brain,the expression of GLUT1 reached the maximum at hypoxia,with significant difference(P<0.05).With the decrease of oxygen concentration,the expression of GLUT1 also decreased,but it was still significantly higher than normoxia(P<0.05).In liver,the expression of GLUT1 in hypoxia 、 semi-asphyxia and asphyxia was significantly higher than normoxia(P<0.05).The expression level of GLUT1 in heart increased with hypoxia stress,and was significantly higher in hypoxia,semi-asphyxia and asphyxia than normoxia(P<0.05).This study clarified the temporal and spatial expression characteristics of GLUT1 gene structure characteristics of silver carp and its response to hypoxia stress,enriching the research data of hypoxia response related genes of freshwater fish.