| Blunt snout bream（Megalobrama amblycephala）is an important herbivorous freshwater farming economic fish in China.Blunt snout bream is widely loved by the Chinese people and has been successfully promoted on a large scale.Compared to other carps,blunt snout bream has the characteristics of intolerance to hypoxia.In the fact,high temperature,hypoxia and other environmental factors often restrict the growth and survival of blunt snout bream at the large-scale intensive breeding process,which seriously restricts its breeding benefits and brings huge economic losses to the breeding industry.Therefore,The shortcoming of blunt snout bream is sensitive to changes in oxygen content,which has always been the focus of our research.The gills are important respiratory organs of fish and are important tissues for hypoxia research.In order to explore the molecular mechanism related to hypoxic response,this study focused on three hypoxic regulation-related genes HIF-1α,HIF-2α,and TET1,and the main research object was F5juveniles of blunt snout bream.the research organization is the blunt snout bream gill.Explored the functional structure of TET1,the spatiotemporal expression pattern of each stage of the embryo,Differential expression in embryo and juvenile tissues under acute hypoxic stress conditions;And different expression patterns of HIF-1α,HIF-2α,and TET1 in response to temperature and hypoxia and their effects on gill remodeling.It provides a basic data reference for the study of the molecular mechanism of hypoxia regulation in blunt snout bream.The main results were as follows:（1）Ten eleven translocation 1（TET1）is a 5–methylcytosine（5m C）hydroxylase,Participates in hypoxic reactions and plays an important role in the hypoxic response process.In order to study the functions and expression characteristics of TET1 gene in blunt snout bream,we first cloned the TET1 gene of blunt snout bream,through sequence analysis,protein secondary structure prediction,and Phylogenetic tree construction to understand basic bioinformatics analysis,we can understand the sequence structure characteristics of TET1 gene;the whole mount in situ hybridization（WISH）and q RT-PCR technique are adopted to analyze the expression of embryos and tissues and study the gene response under acute hypoxic stress.The results of sequence analysis show that TET1 gene is 5526 bp in length,encoding 1841 amino acids;it mainly contains a catalytic domain and a DNA binding domain.The q RT-PCR analysis results demonstrate that TET1 gene is extensively expressed in tissues of blunt snout bream,especially with high-degree expression in its brain.During embryonic development,the TET1 gene has been expressed since the fertilized egg,and the expression during embryonic development has a certain degree of fluctuation.It remains at a high level from 20 to 44 hours（20-44 hpf）after fertilization,and the expression level reaches a maximum at 36 hours after fertilization.The WISH analysis results suggest that TET1 gene detected relatively weak signals in the early stages of fertilized egg development（12hpf）,and the signals gradually increased in the middle and late stages of development（24 and 36hpf）,especially with expression in the head.QRT-PCR is used to detect the expression of TET1 in acute hypoxia.The results manifest that the expression of TET1 gene is significantly increased in gill,spleen and other tissues（P<0.01）,and significantly decreased in brain,skin,eyes and kidneys（P<0.01）.And in embryos,the relative expression of TET1 gene is significantly higher than that of the control group,especially in the 24 hpf Hypoxia treatment group（P<0.001）.The above results indicate that the TET1 gene is involved in the embryo development and the response to hypoxia stress,and thus enhances its adaptability to hypoxia.（2）In order to study the effects of temperature and hypoxia on the expression of HIF-1α,HIF-2α,TET1 genes and gill remodeling in blunt snout bream.We first set two temperatures（25℃,10℃）,performed tissue expression analysis and gill tissue morphology research using q RT-PCR and tissue sectioning techniques.The q RT-PCR analysis results demonstrate that there are no significant difference in the expression of HIF-1αand HIF-2αat different temperatures under normoxic conditions in the gill tissue,compared with normal temperature 25℃,TET1 expression increased significantly at low temperature 10℃;In other tissues,it was detected that temperature had no significant effect on TET1 expression in brain and kidney tissues under normoxic conditions,in the heart,liver,and spleen tissues,compared with the 25℃normoxic group,the expression at the 10℃normoxic group was significantly up-regulated.The observation results of gill tissue sections show that compared with the normal temperature of 25℃,the interlamellar cell mass（ILCM）between adjacent lamella in the10℃low-temperature control group increased significantly（P<0.01）;similarly,the thickness of the exposed lamella was also significantly increased.Subsequently,we set up hypoxia experiments at two temperatures（25℃and 10℃）for 24 hours to study the morphology of the gill tissues of blunt snout bream and expression impact of HIF-1α,HIF-2α,TET1 genes at different temperatures.The results showed that hypoxia induced gill remodeling;the same time of hypoxia stress,the lamella were exposed less at low temperature（10℃）,and the ILCM content was more.In the gill tissue,HIF-1αexpression level was still not significantly affected by hypoxia treatment for 12 and 24hours.but had significant effects on HIF-2αand TET1 expression;In other tissues,TET1 gene was significantly up-regulated in heart,liver,and spleen tissues after 12 or24 hours of hypoxia stress at 25℃or 10℃（P<0.01）,brain and kidney expressions were up-regulated only at low temperature of 10℃.The above results show that the TET1 gene of blunt snout bream has different response times and degrees of hypoxia,and the tissue expression is specific;HIF-2αmay be more active during the hypoxic response than HIF-1αin the gill tissue of blunt snout bream;and blunt snout bream has the ability to adjust the breathing surface area by changing the gill structure to cope with changes in water temperature and dissolved oxygen.（3）In order to further study the role of HIF-1α,HIF-2α,TET1 in the hypoxia response in gill tissue,we used bortezomib to rescue juvenile larvae under hypoxia stress at a temperature of 10℃.Analysis of gill tissue expression and morphological observation of gill tissue were performed using q RT-PCR,tissue section,and scanning electron microscope techniques.Tissue section and scanning electron microscopy results showed that bortezomib eliminated the reduction of ILCM induced by hypoxia in lamella,suppresses the increase in the length of exposed lamella and the increase in the distance between adjacent lamella.q RT-PCR results showed that bortezomib down-regulated HIF-1αexpression,bortezomib also inhibited the increase of HIF-2αand TET1 induced by hypoxia（P<0.01）,the expression levels of HIF-2αand TET1 were almost down-regulated to the levels under normal oxygen.In summary,it is speculated that the hypoxia-inducible ILCM in blunt snout bream is reduced in the HIF-2α-dependent pathway.This study recuperates the vacancy in the study of the hypoxia response of TET1 gene in blunt snout bream,and provides a new reference for studying the regulation mechanism of gill remodeling. |
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