Molecular Cloning And Expression Analysis Of Hypoxia-inducible Factor-1α And-2α In Trachinotus Blochii

Hypoxia-inducible factors(hypoxia-inducible factors,HIFs)are the most important family of transcription factors in a series of regulatory mechanisms when the organism responds to hypoxic conditions.They can effectively sense the decrease of intracellular oxygen concentration and further regulate various genes.Hypoxia-inducible factors are closely related to the growth and development of organisms and the pathogenesis of some diseases.1.The full genomic sequences of HIF-1α and HIF-2α gene from Trachinotus blochii were isolated.The nucleotide sequence of Trachinotus blochii HIF-1α was 3653bp,including 263bp in 5’-untranslated region,1083bp in 3’untranslated region,encoding a protein of 768 aa residues.The nucleotide sequence of Trachinotus blochii HIF-2α was 3157bp,including 48bp in 5’-untranslated region,886bp in 3’untranslated region,encoding a protein of 740 aa residues.Comparing the amino acid sequences of HIF-1α and HIF-2αobtained with several reported vertebrate HIF-1α and HIF-2α amino acid sequences.The results of comparison showed that the homology of HIF-1α in other fishes was more than 84.4%.The highest homology among them was Seriola dumerili.The homology was 92.6%;the homology of HIF-2α with other fish is between 55.0%and 86.8%,among which the highest is Micropogonias undulatus,the lowest is Esox lucius.2.Real-time fluorescence quantitative PCR was used to detect the distribution of HIF-1α and HIF-2α genes in telencephalon,mesencephalon,cerebellum,hypothalamus,gill,heart,liver,kidney,stomach,intestines,spleen and muscle.It was found that both HIF-1α and HIF-2α gene were expressed in 12 tissues detected,and HIF-1α gene was higely expressed in liver.HIF-2awas highly expression in liver gill and mesencephalon.3.The tolerance experiments of Trachinotus blochii to hypoxic environment were carried out in artificial breeding systems.The dissolved oxygen concentration in the experimental water was gradually reduced from 6.5 mg/L to 0.6 mg/L.Dissolved oxygen above 3.5mg/L,no obvious abnormality.When the dissolved oxygen dropped below 3.5 mg/L,the swimming speed of Trachinotus blochii slowed down,and as the dissolved oxygen concentration further decreased,the swimming speed became slower and slower.When the dissolved oxygen is less than 1.0 mg/L,eating is stopped.Through the study of the resistance to hypoxia of Trachinotus blochii,the asphyxiation point of Trachinotus blochii with a body weight of 5.5g-6.5 g was about 0.8-0.9 mg/L at a water temperature of 27.3 ℃.Compared with most freshwater fish and some marine fishes,Trachinotus blochiihas a higher suffocation point.4.In order to reveal the regulation of HIF-la and HIF-2a gene expression under hypoxia stress conditions,the dissolved oxygen was quickly decreased to 1.0 mg/L by nitrogen gas,and nitrogen and air were provided bya stable speed,so that dissolved oxygen is maintained between 1.0mg/L-1.3mg/L.The larvae of Trachinotus blochii were sampled at 1 hour,3 hours,6 hours,12 hours,and 24 hours,respectively,and used as a hypoxic group.After 24 hours of hypoxic treatment,nitrogen was turned off and air was continuously supplied to restore dissolved oxygen to a normal level.After 24 hours of reoxygenation,samples were taken as a hypoxia recovery group.At the same time,untreated individuals under normoxia were used as the control group.Take 6 parallels in each group.Real-time fluorescent quantitative PCR was used to detect the expression of HIF-la and HIF-2a in the four tissues of heart,liver,whole brain and gill.The experimental results showed that the expression of HIF-la was significantly increased in the four tissues after hypoxia stress.Among them,the heart had a significant increase at 12 hours,and the whole brain and liver began to change significantly at 6 hours.After recovery normal oxygenation,the expression of HIF-1α in heart,liver and sputum decreased significantly,but it was still significantly higher than that before hypoxic treatment.There was no change in the expression of HIF-2a in sputum throughout the experiment.In the whole brain,the expression level in the 6h group was significantly higher than that in the other groups,and there was no significant difference between the other groups.In heart and liver,HIF-2α began to rise in the 3 h group,reached its peak at 6 h,and then began to decline.The expression of HIF-2α in tissues after recovery was similar to that of normoxic group.