Effects Of Hypoxia-inducible Factor-1 (HIF-1) On The Alkaline Environment Adaptation Of Naked Carp In Qinghai Lake And Crucian Carp

Located in the northeastern part of the Qinghai-Tibet Plateau,Qinghai Lake is the largest inland saltwater lake in the plateau.Naked carp(Gymnocypris przewalski)is living in Qinghai Lake with high salt(9ppt),high pH(9.0-9.5)and high altitude(average 3260m).Crucian carp(Carassius auratus)is widely distributedin waters all over the country,and is one of the important edible fish in China.Crucian carp is one of the freshwater fish with the strongest hypoxia tolerance and the only fish that can survive indoors without air pump.Hypoxia-inducible factor-1?(HIF-1?)is generally considered to be the main switch for many hypoxia-inducible responses,which is stable and up-regulated in anoxic environments.The conserved sequence of the hydroxylase recognition site in the ODDD functional domain of HIF-1? in naked carp is: PEMLAP(550-555),which is inconsistent with other vertebrate conserved sequences(LXXLAP,X stands for any amino acid).The 550 th Leu which is conserved in other vertebrate species is replaced by Pro,which may affect the stability of HIF-1? in naked carp,and may participate in the adaptation of the saline-alkali lake environment to naked carp,which perhaps has an alkaline water environment adaptation mechanism different from other fish.To explore the effect of HIF-1 on the adaptation of fish to alkaline environment,we used transcriptome sequencing technology,real-time PCR,Western Blot and flow cytometry to study naked carp in Qinghai Lake and crucian carp as follows:1.Transcriptome sequencing analysisThe captured naked carp were be treated separately.Total RNA was extracted from liver tissues and subjected to transcriptome sequencing using Illumina Hiseq high-throughput sequencing technology,obtained the sequence data base number between 3800973300-6587446200 bp.High quality data by de novo assembly to generate contigs and a single sequence for subsequent analysis.Compared with the NR library,33,785 sequences are similar to the sequences of common carp,and the remaining similar sequences can be found in 14 species,while 17179 sequences can not match to its related species and belong to unknown species;By COG classification,many transcripts of the liver of naked carp in Qinghai Lake have been found to have general function,such as signal transduction mechanism,replication,recombination and repair,post-translational modification,protein conversion rate,molecular chaperone,cytoskeleton and other functions.At the same time,KOG classification also found that many transcripts play a role in signal transduction mechanism,biosynthesis of secondary metabolites,transport and catabolism,and transcription.Transcriptome sequencing comprehensively analyze the differences between different groups at transcriptional level,and lay the foundation for the subsequent research on the naked carp in Qinghai Lake.2.Differential expression analysis of key genesReal-time PCR was performed on key genes based on the results of transcriptome sequencing.The crucian carps were purchased from the Jinhua Bird and Flower Market and divided into a neutral group(pH 7.4,normal saline injection),an alkaline group(pH 9.0-9.5,injection saline),and an alkaline DMOG group(pH 9.0-9.5,injection of DMOG 8 mg / kg).Liver tissues were collected for real-time PCR after treatment.First,the relative expression level of VEGF,a HIF-1 target gene,in different groups indicates that HIF-1? is involved in the response of Qinghai naked carp and crucian carp to alkaline environment.Secondly,it was found that the key genes in the tricarboxylic acid cycle and glycolysis pathway were involved in the adaptation of alkaline environment.The relative expression levels of PDK1 and PFK of alkaline group were significantly higher than neutral group.HIF-1? enhances glycolytic pathway and inhibits tricarboxylic acid by activating PDK1 and PFK expression,which promotes the adaptation of naked carp and crucian carp to high ammonia levels in alkaline environments.Thirdly,it was found that alkaline treatment inhibited the expression levels of GS and GDH,key genes of glutamine pathways,in the naked carp and crucian carp,and activated GPT activity in crucian carp.HIF-1 participates in regulating the response of naked carp and crucian carp to alkaline environment by affecting Gln metabolism.Finally,no significant differences were found in the key genes involved in the treatment of fat metabolism pathways.3.Determination of related physiological and biochemical parameters and analysis of cell proliferation and apoptosisThe analysis of the blood ammonia content found that the alkaline treatment caused the blood ammonia concentration of the crucian carp to rise significantly,while the DMOG treatment group resulted in a significant decrease in the ammonia concentration of the crucian carp in the alkaline environment.The blood ammonia content of naked carp was no significant difference between the two groups,indicating that the ammonia tolerance of the naked carp was stronger than that of crucian carp.The analysis of glutamate pyruvic transaminase(GPT)activity and nicotinamide adenine dinucleotide phosphate(NADPH)synthesis rate showed that the alkaline environment treatment significantly increased the GPT activity of in liver tissues of crucian carp,and the GPT activity of the crucian carp could be significantly increased after DMOG treatment,suggesting that the alkaline environment has an effect on the enzymatic activity of GPT,a key enzyme in the amino acid metabolic pathway.To compare the content of nicotinamide adenine dinucleotide phosphate(NADPH)in the neutral environment group(NN),alkaline environment plus saline group(AN),alkaline environment plus DMOG group(AD),DMOG was used to treat the alkaline environment-induced NADPH of the liver tissue of the crucian carp.The decrease in the rate of synthesis indicates that the effect of HIF-1 on Gln metabolism is different in the response of crucian carp to high ammonia environment,which is consistent with the differential expression analysis of the genes.Ammonia treatment of 293 T cells and CIK cells was performed to examine the role of HIF-1 in high ammonia treatment on cell proliferation and apoptosis.CCK-8 analysis found that the cell proliferation ability was was suppressed by ammonia treatment.Apoptosis was detected by flow cytometry Annexin V-FITC and PI double staining.The results showed that the apoptosis rate of the experimental group treated with ammonia was significantly increased,while 1 mM DMOG treatment decreased the apoptosis rate,suggesting that the stability of HIF-1? reduces the damage of the ammonia to cells.