Preliminary Study On The Effects Of Hypoxia On Physiological And Biochemical Indexes And OrPHD2 Gene Expression Of Onchidium Reevesii

Onchidium reevesii is an amphibious mudflat shellfish,which is mainly distributed in the supratidal zone of the southeast coast of China.It is rich in mineral elements and fat-soluble vitamins,and has high medicinal and edible value.In the stage of tidal inundation,it is deprived of oxygen and often in a temporary anoxic state.However,it maintains normal physiological activities.Therefore,we investigated the survival strategy of suchophyllum tuberculatum to reduce oxygen supply.However,as far as we know,hypoxic tolerance in this species has been poorly studied at both molecular and physiological levels.In order to explore the survival mechanism of sulfosulfurous tumor in hypoxic environment,physiological and biochemical indexes of sulfosulfurous tumor were studied,including energy metabolism indexes: glucose,liver glycogen and lactic acid.Oxidative stress index: catalase(CAT),reduced glutathione(GSH)and malondialdehyde(MDA);Immune indices: aspartate aminotransferase(AST)and alanine aminotransferase(ALT).In this study,the gene OrPHD2 was cloned and bioinformatics analysis was performed to study the expression of hypoxia-inducing genes related to hypoxic stress.1.Effects of hypoxia stress on physiological and biochemical characteristics of Onchidium reevesiiIn order to investigate the effects of hypoxia stress on energy metabolism,oxidative stress and immune function,the biochemical indices of serum and hepatopancreas were measured at 0,4,8,12 and 24 h under hypoxia condition [DO was(2.0 ± 0.2)mg/L].The results showed that among the energy metabolism indexes,serum glucose,liver glycogen,serum and hepatopancreas lactic acid contents increased significantly with the time of hypoxia stress.Among antioxidant indexes,catalase(CAT)activity and reduced glutathione(GSH)content increased firstly and then decreased,and both reached the maximum value at 8 h,which were significantly higher than those in control group(P<0.05).The content of lipid peroxide malondialdehyde(MDA)increased gradually with the passage of hypoxic stress time,and was significantly higher than that of control group(P<0.05).Among the immune indexes,alanine aminotransferase(ALT)and aspartate aminotransferase(AST)showed a trend of fluctuation and increase,and were significantly higher than those in the control group(P<0.05).From 8 h on,the contents of ALT and AST in serum were higher than those in hepatopancreas.The results showed that the hepatopancreas was damaged to some extent.The results show that in order to adapt to the hypoxic environment,the body mainly carries out anaerobic glycolysis metabolism,and hypoxic stress will affect the antioxidant capacity and immune function of the body.2.Cloning and functional analysis of OrPHD2 geneThe full length of OrPHD2 gene was 3066 bp.The 5’-end non-coding region is473 bp,and the 3’-end non-coding region is 412bp;The open reading frame(ORF)is1482bp long and encodes 493 amino acids.The molecular mass is 54.51607 Ku,and the theoretical PI is 7.15.The amino acid composition of this protein is Ser(Serine)and Ala(alanine).No signal peptide,belong to transmembrane protein.The OrPHD2 sequence coding region has a P4 Hc domain at 267-454 AA.There are 55 phosphorylation sites,which can play the role of modifying proteins and exert specific functions.Based on homology analysis and phylogenetic tree construction,it was shown that suphd2 is most closely related to the mollusk,Aplysia californica.3.The effect of hypoxia stress on the expression of OrPHD2 in tuberculus sulphonidumThe expression of gene OrPHD2 m RNA in each tissue of Onchidium reevesii was analyzed.The experimental results showed that OrPHD2 was expressed in various tissues to a certain extent,with more expression in hepatopancreas,mouthparts and muscle,and the highest expression in hepatopancreas.In order to explore the expression of this gene under hypoxia,a hypoxic stress experiment was designed with the hepatopancreas,mouthparts and muscle tissues with high expression levels as the analysis objects.The results showed that the expression of gene OrPHD2 in hepatopancreas,mouthparts and muscle showed a trend of first increase and then decrease.As the hypoxia time continued,the expression levels were highest in the hepatopancreas,mouthparts and muscles,indicating that the hepatopancreas was the most sensitive to environmental stimuli.The expression pattern of OrPHD2 exhibits significant temporal and spatial characteristics,so it is speculated that OrPHD2 plays a role in specific tissues to improve the body’s adaptive capacity during hypoxic stress.4.The effect of hypoxia stress on hypoxia-induced gene expressionHepatopancreas is the tissue where the hypoxic response gene OrPHD2 first responds to changes in oxygen concentration.Therefore,we analyzed the expressions of hypoxic inducible genes glycogen phosphorylase(GP),glutathione peroxidase(GPx)and heat shock protein 70(Hsp70)in tumulus chlamydia hepatopancreas.The results showed that with the time of hypoxia stress,the expression level of glycogen phosphorylase(GP)m RNA was significantly up-regulated(P<0.05),indicating that the gene was transcriptional activated by hypoxia stress stimulation.The m RNA expression levels of glutathione peroxidase(GPx)and heat shock protein 70(Hsp70)increased firstly and then decreased,and both reached the maximum value at 8 h(P<0.05),suggesting that hypoxia can activate GPx and Hsp70,and inhibit them to some extent.Based on the analysis of physiological and biochemical indexes in this paper,it was found that the expression of GP showed an opposite trend to the change of liver glycogen,while the change trend of GPx and Hsp70 was similar to that of GSH and CAT.The gene expression level verifies the biochemical index level,which indicates that gene transcription is very important and closely related to protein expression.