Effects Of Hypoxia Stress And Molecular Response In Scapharca Broughtonii

The ability of hypoxia tolerance and adaptive regulation are very important for aquatic animals,especially for species with poor mobility.Previous studies have shown that Scapharca broughtonii has strong hypoxic tolerance.Firstly,the survival of seven species of marine shellfish under different dissolved oxygen(DO)stress was compared.Five tissues of S.broughtonii were observed under 0.5 mg/L DO stress at different time.Illumina Hiseq sequencing platform was used to sequence the hemolymph of S.broughtonii under different concentrations of DO stress.The differentially expressed genes and pathways responding to hypoxia stress were excavated.Three key genes in the hypoxia inducible factor(HIF)signaling pathway were cloned and their gene structure and expression characteristics under hypoxia stress were analyzed.Finally,we preliminarily established RNA interference technology of S.broughtonii,and explored the regulation of HIF-1? on related target genes in HIF signaling pathway of S.broughtonii.The study preliminarily clarified the hypoxic tolerance of S.broughtonii and the response of organism to hypoxic stress at the morphological and molecular levels,which enriched the related research contents of marine shellfish in this field.The results are as follows:(1)The survival and mortality of seven marine shellfish species,S.broughtonii,Cyclina sinensis,Protothaca jedoensis,Mactra veneriformis,Meretrix Cusoria and Ruditapes philippinarum,under DO of 0.5 mg/L,2.5 mg/L and 4.5 mg/L were compared.The mortality rate was calculated and the half lethal concentration was calculated.The results showed that the mortality rate of 0.5 mg/L DO experimental group was significantly higher than that of 2.5 mg/L and 4.5 mg/L experimental group;under 0.5 mg/L stress for 4 hours,the mortality rates of S.subcrenata,Cyclina sinensis and Protothaca jedoensis in experimental group were 55%±3.2%,48%±2.2% and 45%±5.8% respectively,which were significantly higher than those of S.broughtonii,S.subcrenata,Cyclina sinensis and Protothaca jedoensis.After 9 days of stress,the mortality rates of S.broughtonii,C.sinensis,P.jedoensis,M.veneriformis,M.Cusoria and R.philippinarum were 44%±2.2%,48%±1.2%,65%±2%,68%±4.7%,74%±3.8%,88%±4.2% and 100%.At the same time,the LC50 of clam treatment group was significantly lower than that of C.sinensis,P.jedoensis,M.Cusoria and R.philippinarum.3 days of treatment,there was no significant difference between S.broughtonii treatment group and M.veneriformis treatment group.Tissue sections of gill,mantle,axe-foot liver,pancreas and closure muscle showed that there was no significant change in muscle structure of axe-foot and closure muscle of C.quinquefasciatus under DO stress of 0.5 mg/L for 120 hours,while the other three tissues showed more obvious tissue damage at 120 hours.After 24 hours of treatment,the structure of gill filament began to change,mainly as the number of gill filament cilia increased,the contact area between gill filaments increased,the gill filament cavity expanded,the connective tissue in the gill filament cavity thinned,and the proportion of red blood cells in the cavity increased.At 72 hours,the proportion of red blood cells decreased,the proportion of white blood cells increased,and the gill filament cavity began to contract again.The proportion of cells increased significantly,the proportion of white blood cells decreased significantly,and began to decrease significantly at 72 hours.The phenomenon of cell fusion began to appear gradually in the connective tissue of mantle.After 120 hours,the cell fusion of connective tissue of mantle intensified,and the connective tissue began to loosen and disorder.After 24 hours of treatment,the proportion of red blood cells in hepatopancreas tissue began to increase and cell fusion appeared.At 72 hours,the proportion of red blood cells decreased,cell fusion intensified,and hepatopancreas tissue began to loosen.(2)A total of 185 366 unigenes were obtained from the comparative transcriptome sequencing of the three hemolymph RNA samples.Among them,the number of homologous sequences in each species was compared by NR library.The 46,123 unigenes annotated were compared with 935 different species.KEGG Pathway differential enrichment analysis showed that 2040 differentially expressed genes were enriched to 153 Pathways.These signaling pathways were involved in major metabolic pathways and signal transduction pathways,including PI3K-AKT signaling pathway,Carbon metabolism,Fatty acid biosynthesis,and MAPK signaling pathway.Way and HIF-1 signaling pathway are the main pathways related to immunity,metabolism and cell proliferation.The results of fluorescence quantitative validation of 10 differentially expressed genes showed that although the results of these qRT-PCR validation were somewhat different from those of RNA sequencing,the downward and downward trend was the same,which verified the accuracy of transcriptome sequencing analysis.(3)Based on the fragments obtained from transcriptome,the full-length sequences of HIF-1a gene(SbHIF-1a,GenBank No.MH936548),HIF-1? gene(SbHIF-1?,GenBank No.MH936549)and ENO-1 gene(SBENO-1,GenBank No.MK575044)were cloned by RACE and qRT-PCR.The expression characteristics of HIF-1a gene under different experimental conditions were studied.The full length of SbHIF-1a,SbHIF-1? and SBENO-1 genes were 2741 bp,3110 bp and 2411 bp.Three ORFs encoded 711,717 and 603 amino acid residues.The predicted molecular weights of protein were 80.8 kDa,78.4 kDa and 66.5 kDa,respectively.The theoretical isoelectric points were 5.57,6.63 and 5.29.It was predicted that there were two glycosylation sites and a proline hydroxylase binding site LxxLAP.SbHIF-1? gene contains one HLH domain,two PAS domains and one PAC.SBENO-1 gene contains one Enolase-N domain and one Enolase-C domain.The evolutionary tree constructed by the Maximum likelihood model of Mega 6.0 software shows that the molecular evolutionary status of the three genes is basically consistent with their biological taxonomic status.The expression and distribution of SbHIF-1?,SbHIF-1? and SBENO-1 genes in hemolymph,Gill,adventitia,axopod,adductor muscle and hepatopancreas were slightly different by qRT-PCR.The relative expression of SbHIF-1? gene in the six tissues was 11.63 times higher than that in hepatopancreas,including hemolymph,Gill,adventitia,axe foot,adductor muscle and hepatopancreas,and the expression level in hemolymph was 11.63 times higher than that in hepatopancreas.The difference was significant(P < 0.01).The relative expression of SbHIF-1? gene in the six tissues was higher than that in the hepatopancreas,hemolymph,envelope,Gill,hepatopancreas,axe foot and adductor muscle,and the expression of SbHIF-1? in hemolymph was the highest,which was 12.16 times higher than that in hepatopancreas.The difference was significant(P < 0.01).The relative expression of SBENO-1 gene in six tissues was higher than that in Gill,hemolymph,hepatopancreas,adventitia,adductor muscle and axe foot.Compared with axe foot,SBENO-1 expression in Gill was the highest,which was 14.23 times higher than that of axe foot.There was a significant difference between the two groups(P < 0.01).The expression characteristics of three genes in six tissues with DO of 0.5 mg/L,2.5 mg/L and 4.5 mg/L under hypoxia stress are as follows: The relative expression of the three genes in the 0.5 mg/L experimental group was significantly higher than that in the 2.5 mg/L and 4.5 mg/L experimental group.And the relative expression in each tissue was significantly higher than that in the control group after a certain treatment time,especially in hemolymph and gill.The expression of SbHIF-1? in haemolymph increased gradually,while that in other five tissues increased first,then decreased and then increased.In haemolymph,the expression of SbHIF-1? after DO was 0.5 mg/L stress for 4 hours was significantly different from that in control group(P < 0.01).After 64 hours of stress,the expression of SbHIF-1? reached the highest level,which was 519.43 times higher than that in control group.The change trend of SbHIF-1? was more obvious in 0.5 mg/L and 2.5 mg/L hypoxic stress groups.The change trend of SbHIF-1? expression was first decreased and then increased.At 8 h,the relative expression of SbHIF-1? was significantly different from that of the control group.At 36 h,the change reached the maximum,then decreased,and the significant level reached the extremely significant level(P < 0.01).Overall,SbHIF-1? expression in gill,hemolymph and hepatopancreas reached a significant level(P < 0.01).The expression of SbHIF-1? was more significant than that of other tissues.At the lowest level,the expression of F-1beta in the control group was 24 times and 19.43 times respectively.Compared with other tissues,the expression of SBENO-1 in haemolymph was more positive,and the highest value was 89.25 times of that in control group.The overall trend of SBENO-1 expression was first increased with the increase of stress time,and then decreased gradually after 24 hours.The expression of SBENO-1 in gill was similar to that in haemolymph,and the change trend of SBENO-1 increased,then decreased and then increased also reflected the axe and foot.The maxima of SBENO-1 in the 0.5 mg/L experimental group were 6.98,32.31 and 12.69 times higher than those in the control group(P < 0.01).In this study,the gene structure,temporal and spatial expression characteristics and response to hypoxia stress of SbHIF-1a,SbHIF-1? and SBENO-1 were identified,which enriched the research data of HIF pathway genes in marine shellfish.(4)The expression of SbHIF-1a and its target genes,nitric oxide synthase(NOS),transferrin(TF),erythropoietin(EPO),heme oxygenase(Ho)and enolase(ENO),were detected by dsRNA interference.The results showed that SbHIF-1a gene began to decrease at 24 hours after interference,and the relative expression of SbHIF-1a gene decreased significantly at 48-72 hours(P < 0.05).The decrease lasted until 96 hours,and then the gene expression began to reverse.After inhibited expression of SbHIF-1a,the expression of SbHIF-1? gene was significantly up-regulated from 48 h to 72 h(P < 0.05),suggesting that the expression of two subunits of HIF may be mutually inhibited.Compared with the control group,the expression of target genes in the experimental group decreased significantly after 48-96 hours of SBHIF-1a interference(P < 0.05).