Molecular cloning, characterization and expression analysis of a catalase gene in Paphia textile
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Molecular cloning, characterization and expression analysis of a catalase gene in Paphia textile WU Xiangwei1, 2, LI Jiakai1, TAN Jing1, LIU Xiande1* 1 Fujian Provincial Key Laboratory of Marine Fishery Resources and Eco-environment, Fisheries College, Jimei
University, Xiamen 361021, China 2 Animal Science and Technology College, Yunnan Agricultural University, Kunming 650201, China
Received 26 May 2015; accepted 3 December 2015 ©The Chinese Society of Oceanography and Springer-Verlag Berlin Heidelberg 2016
Abstract
Catalase is an important antioxidant protein that can protect organisms against various forms of oxidative damage by eliminating hydrogen peroxide. In this study, the catalase cDNA of Paphia textile (PtCAT) was cloned using RTPCR and rapid amplification of cDNA ends (RACE). PtCAT is 1 921 bp long and consists of a 5′-UTR of 50 bp, a 3′UTR of 349 bp, and an ORF of 1 542 bp that encodes 513 amino acids with a molecular weight of 58.4 kD and an estimated isoelectric point of 8.2. Sequence alignment indicated that PtCAT contained a highly conserved catalytic signature motif ( 61 FNRERIPERVVHAKGAG 77 ), a proximal heme-ligand signature sequence (352RLFSYSDP359), and three catalytic amino acid residues (H72, N145, and Y356). PtCAT also contains two putative N-glycosylation sites (34NKT36 and 437NFT439) and a peroxisome-targeting signal (511AQL513). Furthermore, PtCAT shares 53%–88% identity and 29%–89% similarity with other catalase amino acid sequences. PtCAT mRNA was present in all tested organs, including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, but its expression was highest in the digestive gland. High-temperature-induced stress produced two expression patterns of PtCAT mRNA: first, an initial up-regulation followed by a down-regulation in the heart, digestive gland, and gonad and, second, consistent down-regulation in all other organs. These results demonstrate that PtCAT is a typical member of the catalase family and might be involved in the responses to harmful environmental factors. Key words: Paphia textile, catalase (CAT), cloning, sequence analysis, expression analysis, high temperature stress Citation: Wu Xiangwei, Li Jiakai, Tan Jing, Liu Xiande. 2016. Molecular cloning, characterization and expression analysis of a catalase gene in Paphia textile. Acta Oceanologica Sinica, 35(8): 65–73, doi: 10.1007/s13131-016-0829-6
1 Introduction In organisms, a respiratory burst is defined as high oxygen consumption induced by phagocytes to destroy invading microbes via the innate immune system. During this process, mass reactive oxygen species (ROS), including the superoxide anion (O2·–), hydrogen peroxide (H2O2), and hydroxyl radicals (OH·), are converted from molecular oxygen to directly kill foreign invaders (Lee and Söderhäll, 2002; Hooper et al., 2007). However, the excessive production or accumulation of ROS in cells can cause cellular damage and immune dysfunction by peroxidizing cellular proteins, nucleic acids and lipids, deactivating enzymes, and deregulating redox
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