The Studies On Cloning And Expression Of AmphiCAT In Branchiostoma Belcheri Tsingtauens

H₂O₂ is one of the reactive oxygen species in organisms. Excessive H₂O₂ is harmful for almost all the components of cells, so rapid and effective removal of H₂O₂ is of essential importance for aerobically prokaryotes and eukaryotes. Catalase is an active enzyme which widely exists in all kinds of organisms. Early studies of catalase are mainly concentrated in the chemical characteristics. With the rapid development of molecular biology and gene engineering, more and more scholars means to study the structure and function of catalase and its gene by molecular techniques. Amphioxus belongs to the Cephalochordata. It is an important marine model organism to research vertebrate origin and evolution. The taxonomic status of amphioxus is special. With human destruction of its habitat and pollution of marine environment in recent decades,its resources decreased so sharply that it became endangered species. Catalase can delay aging and improve the immunity of animal body by eliminating the oxygen free radicals. In this paper, we cloned amphioxus catalase（CAT）, analyzed the gene sequences and protein sequences, carried out homology comparison with other species,and constructed the phylogenetic tree. We speculated some properties of the protein by using bioinformatics methods. We did preliminary research on the function of catalase in amphioxus by using in situ hybridization and real-time quantitative polymerase chain reaction（PCR） technology. This research can provide a certain theoretical basis for further studies of amphioxus CAT structure and function, also the disease resistant and disease prevention of amphioxus.In this study, the full-length cDNA of CAT named AmphiCAT（GenBank accession No. KU058636） was cloned and characterrized in amphioxus by using rapid amplification of cDNA ends（RACE）. The full-length cDNA of AmphiCAT was 2640 bp,including a 981 bp 3’untranslated region（UTR）, a 126 bp 5’-UTR and a 1533 bp open reading frame（ORF） encoding 510 amino acids with predicted molecular weight of57.85 kDa. The deduced amino acid sequence of AmphiCAT cDNA had characteristic features of catalase family such as catalytic site motif（HFNRERIPERVVHAKGHGA）and heme-ligand signature motif（RLFSYSDTH）. Using biological software, the protein was analyzed. The results showed that the protein had no signal peptide, and itwas not a secreted protein. Structure prediction showed that amphioxus catalase belonged to clade3 branch of monofunctional catalase. Phylogenetic analysis suggested that amphioxus had close relatives with mollusks, and its classification status was basically consistent with the traditional classification.The primers were designed by using cDNA sequence as template. PCR reactions were carried out by using amphioxus genome as template. Approximately 5000 bp sequence fragment was obtained after splicing. We blasted the gene sequence in NCBI,and speculated that it may contain four introns.In situ hybridization results showed that AmphiCAT transcripts were most abundant in hepatic caecum, intestine, skin, gonads and notochord. High expression in the hepatic caecum, intestine and skin may be related to the fact that these organs were exposed to the external environment such as heavy metals and pathogenic bacteria.Quantitative real-time PCR results showed that the expression of catalase was influenced by heavy metals. When the concentration of chromium ion is 10mg/L, the expression of catalase increased obviously. It may be related to the interaction between enzyme thiol and heavy metals. It also implied that the immune of amphioxus was related to catalase. Although catalase in amphioxus was studied, the immune mechanism is not very clear and should be further studied.