Expression Of Smad Genes In Hyriopsis Cumingii And Exploration Of Wound Repair

TGF-?/Smads signaling pathway plays an important role in early embryonic development,tissue repair,cell proliferation,differentiation,apoptosis and migration,endocrine and immune processes.Studies have shown that TGF-?s are also closely related to extracellular matrix deposition and are one of the main factors affecting wound healing and scar formation.Hyriopsis cumingii is an excellent freshwater pearl oyster.Because insertion and nurturing of pearl caused serious damage to the mussel and affected the quality of pearl breeding,resulting in great economic losses.Therefore,the study of TGF-?/Smads signaling pathway of H.cumingii will be conducive to understanding the immune mechanism of shellfish and provide a theoretical basis for its healthy breeding.1.The specific primers were designed based on the screened fragments of HcSmad3 and HcSmad4 gene from the transcriptome library of H.cumingii hemocytes in our laboratory.Complete cDNA of HcSmad3 and HcSmad4 genes were obtained by used RACE PCR technique.The length of 5'-untranslated region(UTR)of HcSmad3 was 162 bp,the size of 3'-UTR was 633 bp,including two termination signal sequences(AATAAA)and a poly A tail.The open reading frame(ORF)of the HcSmad3 was 1257 bp.It was predicted to encode a polypeptide with 418 amino acids.The theoretical isoelectric point and molecular weight of predicted HcSmad3 protein was 6.09 and 47.28 kDa,respectively.The full length of HcSmad4 cDNA sequence was 2543 bp,consisting of a 5' UTR of 119 bp and a 3' UTR of 876 bp,the length of ORF was 1548 bp,which encoded 515 amino acids.The theoretical isoelectric point and relative molecular weight of HcSmad4 are predicted to be 6.90 and 56.73 kDa,respectively.They both harbored two conservative domains which were MH1(Smad3:29-129aa;Smad4:18-138aa)and MH2(Smad3:222-394aa;Smad4:287-513aa)domains.They also had both NLS(Smad3:39-45aa;Smad4:31-49aa)and NES(Smad3:359,362,365,367aa;Smad4:137-144aa).2.The transcripts of HcSmad3?HcSmad4 and HcSmad5 genes were detected in different tissues of H.cumingii by real-time fluorescent quantitative PCR.The resultsshowed that HcSmad3,HcSmad4 and HcSmad5 were expressed in all detected tissues of H.cumingii.The highest expression was in the muscle,lower in the hepatopancreas,mantle and gill,and lowest in the hemocytes.3.The results showed that the mRNA expression of HcSmad3 in hemocytes was significantly up-regulated compared with blank group at 3 h after A.hydrophila and PGN stimulation,and then these restored to the original level.In hepatopancreas,the expression of HcSmad3 was significantly increased compared with blank group at 6,12 and 24 h after A.hydrophila stimulation,and these decreased to the original level at48 h post-injection.However,the expression of HcSmad3 had no obviously change after PGN stimulation.After stimulation by A.hydrophila,HcSmad4 was significantly up-regulated at 6 h compared with the control group in hemocytes,and the highest expression was found at 48 h in hepatopancreas.4.HcSmad4 mRNA levels were significantly increased post wounding,and the expression of downstream target gene of Smad4,such as HcMMP1,HcMMP19,HcTIMP1 and HcTIMP2 were up-regulated to a certain extent.Whatever knocked down HcSmad3/4 or SIS3 treatment,the expression levels of these genes displayed a significantly down-regulated tendency compared with the wound group.5.Histological evaluation suggested that Smad3-knockdown or SIS3 treatment was accelerated wound healing,and then Smad4 knockdown delayed the process of wound healing in mussels.6.The recombinant plasmids HcSmad3-PET32 and HcSmad4-PET28 were constructed.HcSmad3 protein only existed in inclusion bodies,but HcSmad4 protein could be expressed in supernatant and inclusion bodies.Polyclonal antibodies against HcSmad4 were obtained by immunizing Japanese experimental white rabbits.