| Hyriopsis cumingii is one of the important freshwater mussel in China.TGF-βmediated signaling pathway plays an important role in embryonic development,tissue and organ formation,wound repair and immune response regulation.In this study,TGF-β and I-type receptor(TGF-βRI)gene from Hyriopsis cumingii was cloned and characteristed.The results showed that the full-length cDNA sequence of TGF-β was 2850 bp,5 ’ UTR(non-coding region)was 211 bp,3’ UTR was 1457 bp,the open reading frame was 1182 bp,which encoded 393 amino acids.Predicted TGF-β protein comprised a signal peptide of 25 aa,a TGF-β domain(294-393).The full length cDNA of TGF-RRI gene was 2017 bp,including a 5 ’ UTR 80 bp,3’ UTR383 bp,and the open reading frame(ORF)was 1554 bp and encoded 517 amino acids.Predicted TGF-βRI protein comprised a signal peptide(1-22aa),a membrane outer region(23-127aa),a transmembrane region(128-150aa),and an intracellular region(151-517aa).The intracellular region contained the TGF-β I receptor characteristic sequence GS domain and the serine/ threonine kinase catalytic domain.The sequence comparison analysis showed that the homology of TGF-β of the Hyriopsis cumingii with the mammalian TGF-β was 30%.The predicted TGF-β mature protein was composed of four β-fold and two-α helix,which was similar to the spatial structure of human.The homology of HcTGF-βRI with Danio rerio and Mus musculus was 63%.Real-time quantitative PCR results show that the TGF-βRI were expressed in hemocytes,mantle,muscle,gill and hepatopancreas from healthy mussels.The highest expression was in gill,and lowest expression was in hemocytes.The expression of TGF-βRI was up-regulated at 3 and 6h in hemocytes(p<0.05),and at 6,12 and 24 h in hepatopancreas after Aeromon hydrophila challenged(p <0.05).After peptidoglycan stimulation,the expression of TGF-βRI was up-regulated at 3h in hemocytes and and 24 h in hepatopancreas(p <0.05),and then it returned to the initial level at 48 h,respectively.Moreover,the mRNA expression of TGF-βRI was up-regulated(p <0.05)at the first,third,5th,and 10 th day after wounding,and it restored to the initial level at 15 th day.The maturepeptide encoding sequence of HcTGF-β and the intracellular segment sequence of HcTGF-βRI was inserted into the pET-30 a and pET-32 a plasmid,respectively.The TGF-β recombinant was transferred to Top10 clone strain and BL21(DE3)expression strain,HcTGF-βRI recombinant was transferfromed Ecoli DH5α and DE3,and then transformed bacteria were induced by IPTG,recombinant protein were purified by affinity chromatography(Ni-IDA resin),soluble TGF-β protein was obtained and protein concentration was 0.318 mg/ mL by Bradford method.Recombinant HcTGF-βRI protein was presented in the form of inclusion bodies,the purified protein concentration was 0.5mg/ml.Recombinant TGF-β and TGF-βRI were used to preparated polyclonal antibody after injection into New Zealand white rabbit.Western blot analysis showed that the antibodies against TGF-β and TGF-βRI were highly specific.The antibodies were used to detect the distribution of TGF-βRI protein in hepatopancreas,hemocytes,gill,muscle,mantle and the expression of TGF-βRI protein in the mantle tissue after wounding,the results showed that it is mainly expressed in hepatopancreas and hemocytes,The protein expression level of TGF-βRI was increased accompany with wounding time.After treatment by inhibitor SB431542,the downstream gene Smad3,Smad4,Smad5 of TGF-βsignaling pathway was slightly up-regulated at most time points and down-regulated at a few time points compared with the wound group,while MMP1 gene was significantly down-regulated at the 3d and 5d.MMP19 gene was down-regulated significantly at day 10,and TIMP1 was slightly down-regulated,but there was no significant difference compared with wound group.TIMP2 was significantly down-regulated at day 1 and day 3,and then recovered to the level of wound group. |
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