The Research On The Molecular Mechanisms Of Hypoxia Response In Aurelia Sp.1

A large of problems in marine coastal ecosystem are caused by hypxoia zonesand the blooms of giant jellyfish in estuaries, bays and coastal waters. Compared withother marine organisms, most jellyfish frequently can appear in oxygen minimumzones (MOZ) and has the hypoxic tolerance. However, there are few studies on themechanisms of hypoxia tolerance and response in jellyfish, especially lack of thereports on the molecular regulating-mechanism. Obviously, studies of the relationshipbetween hypoxia and jellyfish blooms must be well performed for a betterunderstanding of the mechanisms of hypoxic tolerance and response.In this study, Aurelia sp.1, frequently emerging in China coastal water, wascultivated in the hypoxic system to reveal the molecular mechanisms of hypoxictolerance and response by detecting the physiological activity, gene expression andmetabolite content based on the hypoxia inducible factor (HIF). Major conclusions arelisted as follows:Firstly, the HIF gene was confirmed to exist in Aurelia sp.1.Based on the partial HIF nucleic acid sequence in the RNA-seq annotation resultof Aurelia sp.1, the full-length cDNA sequence of HIF-1α gene was obtained byreverse transcription PCR (RT-PCR) and rapid amplification of cDNA end (RACE),which contained2,466bp nucleic acids coding675amino acids. The analysis ofHIF-1α protein functional domains showed that there were five conserved functionaldomains including basic Helix-Loop-Helix (bHLH), Per-Ant-Sim (PAS),amino-terminal oxygen-dependent degradation domains (NODD), carboxy-terminaloxygen-dependent degradation domains (CODD) and C-terminal transcriptionalactivation domain (CTAD). Based on the full-length cDNA sequence of HIF-1α, theHIF-1α genomic sequence was obtained by amplification, sequencing and assembling,which consisted of10,739bp nucleic acids. The whole oxygen-dependent degradationdomains of HIF-1α in Aurelia sp.1were similar with other higher animals, while the organization forms of domains and genomic structure were more like ones in loweranimals.Secondly, the HIF-1α was detected in Aurelia sp.1and its expression wasconfirmed to induce by hypoxia environment.About75kDa protein fragment of HIF-1α was obtained by the recombinantprotein expression system in vitro, and anti-HIF-1α poly-clonal antibody withhigh-titer and specificity was gotten successfully by immunzing rabbits. Then wedetected the HIF-1α in Aurelia sp.1using Western blotting and its expression ofHIF-1α was induced by hypoxia environment.Thirdly, the real-time fluorescence quantitative PCR (RFQ-PCR) method todetect relative gene expression related to hypoxic tolerance in Aurelia sp.1was set upusing α-tubulin gene as the reference one.The degenerate primers were designed to amplify the actin gene of Aurelia sp.1by aligning the conserved actin amino acid sequences of cnidarian. Then the partialcDNA sequence of β-actin gene in Aurelia sp.1was cloned and sequenced, whichcontained849bp nucleic acids. After that, four common housekeeping genesincluding18S rRNA, β-actin, α-tubulin and GAPDH were detected their expressionunder normoxia and hypoxia by using real-time fluorescence quantitative PCR(RFQ-PCR) in order to get a suitable reference gene in studying on the molecularmechanisms of hypoxic tolerance and response. The result indicated that α-tubulingene was more stable under different cultivation conditions than others and could beused as a reference gene to study gene expression in hypoxia through the softwareevaluation. Further, the RFQ-PCR method to detect relative gene expression related tohypoxic tolerance in Aurelia sp.1was set up.Fourthly, we had analyzed the variation trends and interaction mechanisms bydetecting the physiological activity, gene expression of PHD-HIF oxygen sensingsystem related genes, metabolism related genes, HSP70gene and metabolites contentin Aurelia sp.1. The findings are listed as follows:①Aurelia sp.1were suffering hypoxia stress in hypoxic cultivation system(dissovled oxygen0.5mg/L) and then showed strong hypoxic adaptability with the adaptive period of approximately30hours. Because of absence of oxygen incultivation system, the physiological activity of Aurelia sp.1showed to be suppressedthrough the decreasing of the bell contraction rate and increasing of the ralativeexpression of HSP70gene; however, it would gradually return to normal state inmechanism transformation and adjustment after30hours.②The peak value ofrelative expression of PHD-HIF oxygen sensing system and metabolism-related genesall occurred ater18hours, which indicated that the mechanism transformation andadjustment at the molecular level in Aurelia sp.1was most active at that time so as toresponse to hypoxia.③PHD-HIF oxygen sensing system played an important rolein the process of tolerance and response to hypoxia. Our experiment result indicatedthat the relative expression of PHD and HIF-1α gene was stable under normoxia andshowed a trend of first increasing then falling under hypoxia, which supported theregulatory mechanism of PHD-HIF oxygen sensing system. With prolonged time ofhypoxia, the transcriptional expression of HIF-1α gene was up-regulated and HIF-1αprotein was quickly synthesized. Immediately, the HIF-1α protein combined to theenhancer region of PHDs gene and induced the transcriptional expression of PHDsgene.④There was an anaerobic metabolism process depending on lacticdehydrogenase under hypoxia in Aurelia sp.1. The relative expression of LDH geneand concentration of lactic acid both gradually increased with prolonged time ofhypoxia, which indicated that the reversible reaction catalyzed by lacticdehydrogenase was to produce lactic acid.Fifthly, the molecular mechanisms of hypoxic tolerance and response in Aureliasp.1was the shift from aerobic to anaerobic metabolism regulated by PHD-HIFoxygen sensing system.Under hypoxic conditions, the HIF-1α transcription factor was induced byhypoxia and quickly attached to the enhancer regions of the key glycolytic genes toinduce the target genes expression, which provided enough energy to maintain thephysiological activity of Aurelia sp.1by accelerating the glycolytic pathway. Furtherly,the expression of LDH gene that was induced by HIF-1α converted the pyruvate tolactate so as to fulfill the shfit from aerobic to anaerobic metabolism. Sixthly, the large expression of HIF-1α protein under hypoxia was probably dueto the results of co-regulation at the transcriptional and post-translational levels.The transcriptional level regulation of HIF-1α in Aurelia sp.1was demonstratedby the experiment. The analysis of functional domains revealed that HIF-1α ofAurelia sp.1contained the oxygen-dependent degradation domains (ODD), and thePHDs gene found in the RNA-seq was induced by HIF-1α under hypoxia. Thoseevidences mentioned above indicated that there was post-translational level regulationof HIF-1α. Therefore, the large expression of HIF-1α protein under hypoxia wasprobably due to the results of co-regulation at the transcriptional andpost-translational levels.This study provides the scientific basis for clarifying the correlation betweenmarine hypoxia and jellyfish blooms and revealing the key environmental factorswhich cause the jellyfish blooms.