Identifcation And Functionalanalysis Of IGFBP-1and HIF-1α Of Paralichthys Olivaceus

Insulin-like growth factors (IGFs) is necessary for vertebrates growth anddevelopment, and IGFBP-1is an important member of the system. It is not only relatedwith growth closely, but also sensitive to the oxygen concentration, and IGFBP-1is animportant target gene of HIF-1, which induced the IGFBP-1expression to adapt to lowoxygen environment. Japanaese flounder (Paralichthys olivaceus) is one of theimportant economical flatfish and undergoes a dramatic metamorphosis，involvingtransformation from a bilateral pelagic larva to an asymmetrical benthic juvenile withboth eyes on the same side of the body. We have constructed IGF-1and its receptorexpression profiling in the development of the flounder metamorphosis and analysed thepotential role of IGFs in flounder development and larval metamorphosis. However,how IGFBP-1regulated the growth and development of the flounder, what regulatorymechanism of IGFBP-1and HIF-1involved in the hypoxia has not been reported so far.So, we cloned the flounder IGFBP-1and HIF-1α cDNA, and detect the mRNAexpression at different developmental stages, in different organizations and underhypoxic stress, and detect several enzyme changes related to oxygen metabolism, inorder to further study the role of the IGF system in the growth and development of theflounder, and described the molecular mechanisms of how HIF-1α regulate IGFBP-1gene expression in hypoxia. The results are as follows:In this study, we was used rapid amplification of cDNA ends (RACE) technologyto clone cDNA of IGFBP-1and HIF-1α from the liver of Japanaese flounder. TheIGFBP-1cDNA is1070bp, containing a period of69bp5’-untranslated region (UTR)and272bp of3’-UTR, and an open reading frame of729bp, encoding242amino acids.It displayed the highest homology of89%to S. maximu and S. quinqueradiata, andsequence similarities between P. olivaceus and P. favescens, S. alpines, C. carpio, D.rerio, are84%,79%,67%and67%, respectively. The full length cDNA of HIF-1α is 2330bp, containing a period of225bp5’-UTR and110bp of3’-UTR, and an openreading frame of1995bp, encoding664amino acids. The deduced amino acid sequenceof flounder HIF-1α with other fish and mammals such as P. flesus, S. lucioperca, P.fluviatilis, S. salar, C. carpio and H. sapiens homology are90%,83%,83%,71%,62%,53%.In this study, quantitative RT-PCR showed that the highest IGFBP-1geneexpression in flounder liver. It is468.7times as much as the muscle (P <0.05); followedin the stomach, spleen, intestine, gonads, kidney, gill, brain, heart and muscle, nosignificant differences between each other. The result showed certain levels of IGFBP-1transcripts were detected in unfertilized egg, fertilized egg, blastocyst stage and gastrulastage, but it had a very low level from neurula stage to hatching. A relatively high levelof IGFBP-1mRNA was present until3dph, but in the next7dph to20dph, it sharplydecreased and remained a lower level. From23dph, IGFBP-1mRNA graduallyincreased and got to the highest level at29dph when the larvae were just atmetamorphic climax, and it declined visibly at the end of metamorphosis, whichspeculated that the IGFBP-1gene may play an important role in the flounder embryonicand larval development. Semi-quantitative RT-PCR results showed that HIF-1α mRNAis expressed in the flounder embryos, larvae and all tissues which were in detected，andthe expression of HIF-1α gene is sustained high level after7pdh, and high levelexpression in all tissues except liver.Thyroid hormone plays an important role in flounder metamorphosis, and IGFswere signifcantly altered under TH treatment during the P. olivaceus metamorphosis.Therefore, this experiment using quantitative PCR to detect the IGFBP-1mRNAexpression under TH treatment at different time points in flounder metamorphosis. Theresult of this study showed TH-treated larvae exhibited signifcantly higher IGFBP-1mRNA levels than untreated larvae at23,29,36and41dph (1.5-fold to13.3-folddifference, respectively, P<0.05).In this study, we also examined the enzyme activity change, which related withoxygen metabolism under normal oxygen (11.18±0.25)mg L-1,70%oxygen(7.82±0.18)mg L-1,50%oxygen (5.57±0.22)mg L-1for48hours. The result showed thattotal antioxidant capacity in the liver compared with normal condition significantdifference and increased by23.1%to225%, respectively (P<0.05). Lactatedehydrogenase in the liver and muscle increased compared with normal condition increased by13.4%～60.3%, respectively (P<0.05), superoxide dismutase in the liverand muscle increased by14.5%～81.4%, respectively (P<0.05), and hydrogen peroxideactivity has decreased compared with control group. In this study, we also detected theexpression of flounder IGFBP-1mRNA under hypoxic conditions by quantitativeRT-PCR, the results showed that IGFBP-1mRNA expression in the liver has reducedsome degree under hypoxic, and has significant difference (P <0.05) with the normalgroup. In the gills, the expression of IGFBP-1mRNA has also reduced, and hassignificant difference (P <0.05) with the normal group at24h and48h. In muscle, in50%oxygen environment, the expression of IGFBP-1was the lowest for14.9%of thecontrol group at24h, and increased to440.8%of the control group at48h, in70%oxygen group,the level of IGFBP-1expression was17.0%of the control group, andincreased to241.0%of the control group at48h. This study provides an experimentalbasis for exploring hypoxia stress-related gene expression changes, as well as researchflounder physiological changes from the molecular level in low oxygen conditions, andlaid a theoretical foundation for improving the capacity of flounder tolerance tohypoxia.