Cloing,mRNA Expression Analysis Of MPO CDNA And Cytochemical Characterisation For MPO Of Blood Cells In Silurus Meridionalis

The Wright-Giemsa (W-G) and cytochemical staining of enzyme methods were used for study of cytochemical characterisation for myeloperoxidase (MPO) of blood cells in Silurus meridionalis, and compared with peroxidase (POX) staining characterisation of blood cells in tetrapods, cypriniforms and siluriforms. Full-length of MPO cDNA in Silurus meridionalis was cloned and analysis by RT-PCR and RACE, tissue expression and ontogeny of MPO were also studyed by RT-PCR. The regularity on content variation for MPO of hematogenesis and cytochemical characterisation in Silurus meridionalis were made clear, the regularity on the changes of MPO and EPO on phylogeny of vertebrates and phylogenetic station of teleostei MPO gene in POX gene family of vertebrates were summarized, and we acquired tissue and temporal expression pattern on ontogeny of MPO mRNA in Silurus meridionalis. This study could enrich researches on cytochemistry of blood cells in fishes, and provide the data on studies of MPO function in hematopoietic regulation.MPO could represent the sign enzyme in Silurus meridionalis, which is specific to neutrophil. Compared with the cytochemical characterisation for MPO of blood cells in some cypriniforms and siluriforms, we found that activity of MPO in siluriforms is significantly greater than it in cyprinid fishs, thereby we presumed that the relativaly strong ability to resist disease in Silurus meridionalis is related to their higher MPO activity in neutrophils.MPO might be originate from the promyelocyte in Silurus meridionalis, the content of MPO was reduced in the mature neutrophils, and was increased in the metamyelocyte,which indicated that the content increased at first, and then decreased during the granulocytes morphogenesis. Compared with the content of MPO on hematogenesis in Silurus meridionalis and other vertebrates, the results showed that regularity of MPO morphogenesis in Silurus meridionalis was similar to other fishes and mammals. The cytochemical characterisation of MPO in Silurus meridionalis was relatively similar to amphibians, reptiles and mammals, while showing a great difference with avian. So we presumed that the relationship of MPO and EPO may indicate that the two can be complementary or substitute in their function during vertebrate's phylogeny.Full-length MPO cDNA sequence of Silurus meridionalis consists of 3356 nucleotides, including an open-reading frame(ORF)of 2355nt which encodes a section of signal peptide(M'-S24), a section of precursor peptide(E25-P139), a section of light chain(C140-G254) and a section of heavy chain(I255-K784), and 45 nt 5'-untranslated region(5'UTR),956nt 3'-untranslated region(3'UTR). The MPO shares 77% amino acid sequence identity with channel catfish, and 66% with zebra fish. Catalytic residues in the light chain and heavy chain of MPO were conserved among mammals and Silurus meridionalis, the Ca2+ binding motif were also relatively conserved. The peptide has three potential N-glycosylation sites which is insignificant different among mammals and Silurus meridionalis, and the covalent heme linkage residues with the heme group has insignificant different beteen human and Silurus meridionalis. We presumed that catalytic mechanism and activity of MPO protein in Silurus meridionalis may be similar to mammals.Phylogenetic analysis of mammalian peroxidase amino acid sequences (myeloperoxidases, eosinoperoxidases and thyroid peroxidases) and MPO of teleostei showed that MPO gene of teleostei have undergone their own independent process of duplication after teleostei diversification from vertebrate. MPO gene of teleostei and MPO and EPO gene of tetrapods have the common ancestor. We speculated these gene duplication and diversification in tetrapods that created MPO, EPO, lactoperoxidases, and salivary peroxidases postdate the last common ancestor of tetrapods and teleostei. We also speculate that TPO may be the first divided from teleostei POX genen family, followed by EPO, the MPO gene divided and EPO lost with the degradation of eosinophil phagocytosis in teleostei. MPO gene reserved within neutrophils phagocytosis evolution in different teleostei.MPO mRNA of Silurus meridionalis highly expressed in kidney, head kidney, skin and gill, showing abroad expression in tissues. This expression model was according with their function of participating in non-specific immune system. MPO mRNA was first seen at tail bud free stage in embryo of Silurus meridionalis, which was significantly increased when embryo development to the heart apparition, and was highest to prophase of out from membrane.The expression levels decreased slightly to newly hatched larvae stage, and to the pelvic and dorsal fin appeared stages, the expression level slightly higher than the larvae stage, but there were no significant difference among their own. The results indicated that the expression model of MPO in embryo development of Silurus meridionalis was closely related with the development of hematopoietic system.