Gene Cloning, Expression And Function Of MyD88, CAT And GST In Scapharca Broughtonii

The ark shell Scapharca broughtonii is a kind of high protein, low fat food, which has good market prospects and development value. As with other invertebrates, S.broughtonii mainly rely on innate immunity factors of immune defense. However, S.broughtonii has its own particularity, whose haemolymph contains red blood cells. And the red blood cells have been proved not only to transport oxygen but also to play a role in immunity. At present, there are a few immune related genes of S.broughtonii such as manganese superoxide dismutase, ferritin, galectin and big defensin having been studied. Therefore, Studing immune related factors of S.broughtonii will be improtant to enrich its immunology data. In the present study, the complete cDNA of catalase, myeloid differentiation factor 88, glutathione S-transferase in ark shell S. broughtonii(named SbCAT, SbMyD88, SbGSTμ) was cloned by RT-PCR and rapid amplification of cDNA ends technique and carried out certain immunological studies, respectively.The full-length of c DNA of catalase contained 2181 bp.The cDNA consists of a 5’ untranslated region(UTR) of 96 nucleotides, the 3’ UTR of 654 bp, and an open reading frame(ORF) of 1431 bp, encoding 477 amino acid residues with 54 kDa predicted molecular weight and the theoretical isoelectric point of 8.03. SbCAT amino acid sequence with the other animals are higher consistency,with 68%-96%. The deduced amino acid sequence of SbCAT has characteristic features of catalase family such as the catalase active site(61FNRERIPERVVHAKGAG77),the catalase heme-ligand signature motif(351RLFSYPDTH359) and the three catalytic amino acid residues of His-72, Asn-145 and Tyr-355. In addition, SbCAT also has the conservative heme-binding pocket and NADPH binding sites. Quantitative real-time PCR(qRT-PCR) method was used to analyze the SbCAT mRNA expression characterization in tissues of normal ark shells. The results showed that SbCAT mRNA detected expressed in six kinds of organizations and which was higher in the tissues of mantle and lower in the tissues of hepatopancreas and haemocyte. After Vibrio anguillarum and Staphylococcus aureus challenged, SbCAT expression was rather low in mantle with V.anguillarum challenged, and significant up-regulated in other tissues.The full-length of cDNA of SbMyD88 was 1564 bp, with a 1326 bp open reading frame encoding a protein of 442 aa, with predicted molecular weight 50.2 kDa and the theoretical isoelectric point 5.32. The SbMyD88 contained death domain and Toll/interleukin-1 receptor(TIR) domain which are typical features of MyD88 family proteins. In the TIR domain, three conserved boxes, box 1, box 2 and box 3, were found. According to the comparison with MyD88 amino acid sequences from other species, the putative AA of SbMyD88 was highly consistent with others, of cosistency value 47%-61%. All the sequences were clustered into two main branches in which all MyD88 of molluscs clustered together, while the MyD88 of vertebrate formed the another separate cluster. The expression level of SbMyD88 mRNA was investigated in healthy and challenged ark shells by quantitative real-time PCR. The SbMyD88 gene expression was ubiquitous in all selected tissues. In special, expressed weakly in hepatopancreas and strongly in gill. Compared with that in hepatopancreas, the transcription of the SbMyD88 in gill increased 347.72-fold. SbMyD88 was up-regulated in all tissues after challenge with both V.anguillarum and S.aureus. Those results indicate that SbMyD88 can response against bacteria challenge, which is an important factor in the innate immune response in ark shell.The 1040 bp of cDNA of SbGSTμ included an open reading frame of 648 bp encoding a polypeptide of 216 amino acid residues with a molecular mass of 24.9 kDa and an estimated p I of 8.11. Sequence analysis revealed that the SbGSTμ possessed several characteristic features of μ class GSTs, such as a thioredoxin-like N-terminal domain containing binding sites for glutathione(GSH) and a C-terminal domain containing substrate binding sites. The mRNA expression profiles of SbGSTμ in tissues of foot, gill, mantle, adductor muscle, hemocytes and hepatopancreas analyzed by quantitative real-time PCR(qRT-PCR) suggested the mRNA transcripts of SbGSTμ distributed in all the examined tissues. Importantly, V.anguillarum, S.aureus and CuSO4 challenge resulted in the increased expression of SbGSTμ mRNA with a regular change trend in all examined tissues, indicating SbGSTμ may play an impront role in the immune response process. The optimum pH and temperature for the catalytic activity of the SbGSTμ recombinant protein were 7.4 and 40 °C. In vitro cytotoxicity assays, recombinant protein SbGSTμ can reduce the damage of DNA bases,which BaP causes.