Cloning And Expression Of Hypoxia Inducible Factor-1? Gene From Marsupenaeus Japonicus

Marsupenaeus japonicus has a very delicious meat quality and rich nutrition.It occupies a very important position in the shrimp culture industry in China.Because of its relatively low temperature resistance and strong disease resistance,the area and scale of its culture increase rapidly in recent years,especially in Shandong and other places in the north.With the increase of demand,the density and scale of breeding increased gradually,and the outbreak of disease became more frequent.Coupled with its lower unit production and less market supply,prices have remained high.For aquaculture animals,heavy metals and antibiotics are two common stress factors,which have a great influence on the quality of water and the living environment of aquatic organisms.Heavy metals can cause heavy metal poisoning,produce stimulation,and cause adverse effects on its growth,abnormal development,respiratory metabolism and osmotic pressure regulation.However,a moderate amount of heavy metals,such as metal copper,flow,oxygen supply and cell division for the blood of shrimp.The role of building up.The addition of antibiotics can improve the immunity of the shrimp and increase the resistance to other stress factors such as hypoxia and virus,and a proper amount of antibiotics in the shrimp pond can be used as a way to improve the resistance of prawn itself.Hypoxia-inducible factor(HiF-1)can be considered as a key regulator of the induction of hypoxia gene and the repair of the intracellular oxygen environment.The interaction of oxygen directly with the oxygen receptor is not related to mitochondrial respiration,and the oxygen receptor is in the inactivation state.Oxygen receptor activates a signal when oxygen decreases.After protein phosphorylation and/or oxidation-reduction,the expression of HIF-1/beta is increased and the reoxygenation decreases.Heme containing proteins are involved in oxygen sensing and signaling.Hypoxia or some oxidative stress can provide information for hypoxic induction.Although all cells have the same pathway,each cell has different reaction endpoints.Therefore,hypoxia induces most of the cells to produce glycolysis genes and various vascular factors,but the liver and kidneys induce EPO.Little is known about the mechanism of hypoxia induced gene expression.The expression of HIF-1 in hypoxia is only a phenomenon,and the "killer" behind it needs to be identified.The resulting gene expression seems to increase or decrease oxygen consumption and maintain stable internal environment in order to increase hypoxia tissue oxygen supply.However,more and more bad,glycolysis,mitochondrial metabolism weakened;red blood cells increased,blood thickening;platelets were many,thrombus was easy to form;endothelin,interleukins,Hela cells(uterine cancer cells)had more advantages and fewer disadvantages.How to ensure smooth blood oxygen and improve tolerance under anoxic environment/condition is very important.The above functions of HIF-1 have great potential application in shrimp culture.In this paper,the cDNA sequence of the hypoxic inducible factor gene(HiF-1)of Japanese Penaeus japonicus was obtained by means of common PCR and RACE,and the sequence structure of cDNA,the physical and chemical properties of the encoded protein,the spatial structure and the evolutionary relationship were predicted by the means of bioinformatics.And analysis.Through Real-Time PCR,the distribution of HIF-1 alpha in various tissues,and the changes in the expression of HIF-1 alpha in the hepatopancreas with different concentrations of copper ions and oxytetracycline stress at different concentrations were observed.The 96hLC50 value of Japanese shrimp(Penaeus japonicus)under organic complex copper stress was also obtained.The main results are as follows:1.The cDNA sequence of HIF-1 alpha is full length 3603bp,including the open reading frame(ORF)of 3105bp,the 5 'non coding region(UTR)is 55bp 3' non coding region(UTR)of 442bp,and encodes 1035 amino acids.The sequence contrast results showed that the cDNA sequence of HIF-1 alpha of Penaeus japonicus and the cDNA sequence of Litopenaeus vannamei HIF-1 a was the highest,which was 99%.Through the analysis of the software,the theoretical molecular weight is 113.515kDa,the isoelectric point is 6.36,the instability index(II)is 53.20,the aliphatic index is 77.93,and the signal peptide is free.The total hydrophilic average(GRAVY)value is-0.336,which is a hydrophilic protein.2.Real-Time PCR technique was used to analyze the expression of HIF-1 alpha in the tissues of Marsupenaeus japonicus.The results showed that HIF-1-alpha was expressed in 8 different tissues,such as hepatopancreas.However,there were differences in the expression levels of hepatopancreas,gills,muscles,intestines,eye stalk,stomach,heart and blood in Bazhong.The highest expression level was hepatopancreas and gills,followed by hemolymph.Other tissues such as intestines,muscles,hearts,eye handles and stomach are very low in expression.3.The fluorescence quantitative test of HIF-1 alpha in hepatopancreas showed that the expression of HIF-1 alpha in the hepatopancreas of Marsupenaeus japonicus began to decrease after different concentrations of copper ion stress,and then all up up,and the higher concentration group was up to be more obvious.With the change of time,the expression level decreased first,then increased and then decreased.The expression level of the experimental group was significantly different from that of the control group.At different concentrations of oxytetracycline stress,the expression of HIF-1 alpha in the hepatopancreas of the Marsupenaeus japonicus was up to the beginning,and the higher concentration group was more obvious in the lower concentration group.With the change of time,the amount of expression increased first and then decreased.4,The 96h toxicity test of heavy metal organic complex copper ions in Marsupenaeus japonicus showed that the safe concentration of Cu to the shrimp(average weight 8.43g)of the Japanese sac was 2.61moI/L(0.168ppm).Under the condition of Cu 96h pollution experiment,with the increase of Cu concentration,alpha decreased first and then increased with the increase of Cu concentration in the environment.The results of this study will be helpful to further explore the biological function and application of MT in shrimp Penaeus japonicus,and provide important basic information for the healthy culture of Penaeus japonicus.