Molecular Cloning And Functional Analysis Of Hemoglobin Genes From Ark Shell Scapharca Broughtonii

Scapharca broughtonii is a kind of aquatic animal which is rich in protein and B12,which has good market prospect and economic value.Previous studies have found that S.broughtonii has strong anti-stress ability and red blood cells in hemolymph,which is one of the important characteristics of other shellfish.Hemoglobin is an important component of red blood cells.However,there is very limited research on the hemoglobin of S.broughtonii.In order to further clarify the gene structure and function effect of the hemoglobin,we compare the survival status of S.broughtonii,Chlamys farreri and M.veneriformis in low oxygen stress.We have cloned 3 species of the hemoglobin gene of S.broughtonii and analyzing the characteristics of the expression.Through in vitro expression,the recombinant protein of the two kinds of S.broughtonii hemoglobin was obtained,and those enzyme activity was studied.The study on the enhancement of the cell blood cells of S.broughtonii can enrich the research on the disease resistance and immune mechanism of shellfish,and it is of great significance to the artificial cultivation of shellfish.The semi-death concentration of the cockles is lower than that of the scallop and the four horned clams.In the 3 days,the LC50 of quebecca is only 1.97,while the scallop and the clams are 4.46 and 4.09 respectively.Dissolved oxygen is one of the important environmental factors affecting the survival,growth and energy metabolism of aquatic animals.Shellfish can produce a lot of response mechanisms after low oxygen stress,but there are great differences in the ability to tolerate hypoxia.This experiment through the survival condition,superoxide dismutase(SOD)activity and total antioxidant capacity(T-AOC)in 4.5 mg/L,2.5mg/L and 0.5 mg/L oxygen condition of S.broughtonii,C.farreri,Mactra veneriformis research,and found that the survival rate of S.broughtonii were higher than those of C.Farreri and M.veneriformis,and there was a significant difference between 2.5mg /Land 0.5mg /L group after 1 day(p<0.01).The semi-death concentration of S.broughtonii is lower than that of C.farreri and Mactra veneriformis.In the 3 days,the LC50 of S.broughtonii is only 1.97,while the scallop and the clams are 4.46 and 4.09 respectively.The SOD enzyme activity and T-AOC of gill,mantle and hemolymph have varying degrees of change.But the S.broughtonii group can return to normal levels that with the extension of stress time at 72 h,and C.Farreri and M.veneriformis significantly lower than the control group,meanwhile there were significant differences between the scallop groups(p<0.01).It is shown that S.broughtonii is a higher level of hypoxic tolerance than C.Farreri and M.veneriformis.Hemoglobin being widely distributed in animals,although its important oxygen transport functions has been extensively reported,studies on the immune function of hemoglobin in mollusc are few.Researches on immune of hemoglobin of ark shell S.broughtonii attracted more and more attention.In this study,the hemoglobin c DNAs of S.broughtonii were cloned by EST and RACE methods(named as Sb Hb IIA?Sb Hb IIB and Sb Hb IIC).And the m RNA expression were studied under different conditions.Sequence analysis revealed that the c DNA of Sb Hb IIA was 867 bp in length,including an open reading frame(ORF)of 483 bp,encoding 160 amino acid residues with 17.5k Da predicted protein molecular weight and the theoretical equivalence point of 9.68.The c DNA of Sb Hb IIB was 946 bp in length,including an open reading frame(ORF)of 459 bp,encoding 152 amino acid residues with 17.1 k Da predicted protein molecular weight and the theoretical equivalence point of 5.1.The c DNA of Sb Hb IIC was 916 bp in length,including an open reading frame(ORF)of 447 bp,encoding 148 amino acid residues with 15.8 k Da predicted protein molecular weight and the theoretical equivalence point of 7.9.The amino acid sequence has multiple heme binding sites,and has the characteristic sequence of hemoglobin.Sequence and homology analysis showed that the hemoglobins shared similarity with that of other related species.The results of real-time quantitative PCR(qrt-pcr)showed that there were high consistency in the variation trend of three hemoglobin genes.It can be detected in the foot,gills,mantle,adductor muscle,haemocytes and hepatopancreas,and the fertilized egg to the larvae that developed for 23 days.The expression level of adult hemolymph was the highest,and there was a significant difference between the expression levels of the other five tissues(p<0.01).The expression of the fertilized ovum to 23 d larval stage,SbHb IIA,SbHb IIB and SbHb IIC were all significantly increased from the 12 th day of development(p<0.01),and Up to 200 times reaching than the lowest value.The expression of gene in hemolymph increased first and then decreased after hypoxia stress.The treatment group of 4.5 mg/L,2.5 mg/L and 0.5 mg/L were reached its maximum at16 hours,and the expression level was more than four times that of the control group.After the stimulation of Vibrio anguillarum,the expression of the three hemoglobin genes showed a trend of first rise and then decline,but the maximum expression level was different.The peak of the expression of Sb Hb IIA and Sb Hb IIB appeared at 16 h,while the expression peak of Sb Hb IIC appeared at 32 h.It can be inferred that the hemoglobin gene of S.broughtonii plays a role in the immune response,belonging to an immune factor.Recombinant protein of hemoglobin(r Sb Hb IIB and r Sb Hb IIC)were successfully obtained by prokaryotic expression,and further function analysis indicated obviously that the proteins had very strong phenoloxidase-like activity(PO-like activity)by the response to L-DOPA.The optimal p H and temperature of PO-like active at r Sb Hb IIB was 30 ? and 5.0,respectively,while r Sb Hb IIC optimal p H and temperature was 7.4and 40 ?.Various oxidase inhibitors(ascorbic acid,thiourea,citric acid,etc.)and metal chelate(EDTA)have significant influence on the recombinant protein po-like activity.Metal ions(Fe2+?Zn2+ and Cu2+,etc.)have great difference in their enzymatic activities,Mg2+ and Ca2+ had little effect on po-like activity,while Cu2+ inhibited its enzyme activity,but Fe2+ had a catalytic effect on enzyme activity.The po-like activity of recombinant protein can be recovered from EDTA pretreatment by Fe2+ that was verified by experiments.It could remarkably inhibit growth of gram-negative bacteria V.anguillarum.The hemoglobin has been proved to antibacterial function from the m RNA and protein levels.This paper provides the basic information for the research on the follow-up immune function of quebecca..