cDNA cloning, characterization and expression analysis of the antioxidant enzyme gene,catalase,of Chinese shrimp Fenneropenaeus chinensis
Zhang Q, Li F, Zhang X,et al. Fish Shellfish Immunol. 2008;24(5):584-591. Speaker：Ying-Chun Yeh Advisers：Dr. Nai-Yueh Tien Location：Q004 Date：10/23/2008 Abstract Catalase is an important antioxidant protein that protects organisms against various oxidative stresses by eliminating hydrogen peroxide. The full-length catalase cDNA of Chinese shrimp Fenneropenaeus chinensis was cloned from the hepatopancreas using degenerate primers by the method of 3’ and 5’ rapid amplification of cDNA ends PCR. The cDNA sequence consists of 1892 bp with a 1560 bp open reading frame, encoding 520 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases. The sequence includes the catalytic residues His71, Asn144, and Tyr354. The molecular mass of the predicted protein is 58824.04 Da with an estimated pI of 6.63. Sequence comparison showed that the deduced amino acid sequence of F. chinensis catalase shares 96%, 73%, 71% and 70% identity with that of Pacific white shrimp Litopenaeus vannamei, Abalone Haliotis discus hannai, Zhikong scallop Chlamys farreri and Human Homo sapiens, respectively. Catalase transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill by real-time PCR. The variation of catalase mRNA transcripts in hemocytes and hepatopancreas was also quantified by real-time PCR and the result indicated that the catalase showed up-regulated expression trends in hemocytes at 14 h and in hepatopancreas at 37 h after injection with white spot syndrome virus (WSSV). Introduction Invertebrates do not possess an adaptive immunity based and must rely on innate immunity against invaders. The hemocytes are key cells for invertebrate's innate defense reactions. One important immune defense reaction of invertebrate hemocytes is phagocytosis. During the course of phagocytosis, the host’s NADPH-oxidase is activated which in turn enhances the glycolytic reactions that will increase the consumption of oxygen, and induce the production of reactive oxygen species (ROS). These ROSs can kill foreign invaders efficiently and also play an important role in immune signal transduction. However, the mass accumulation of these reactive molecules in animals will cause serious cell
damage. Catalase (CAT), involved in crustaceans’ innate immune reaction, is an important antioxidant enzyme that is eliminating excessive hydroperoxide and maintaining cellular redox balance. In this study, a full-length cDNA of catalase was cloned from Chinese shrimp Fenneropenaeus chinensis and its expression profile in hemocytes and hepatopancreas was studied after infection with WSSV. Materials and methods 1. 2. 3. 4. 5. 6. 7. Experimental animal and immune challenge RNA extraction and cDNA synthesis Degenerate primer design and strategy of catalase cDNA cloning Rapid amplification of 5’ and 3’ cDNA ends Analysis of nucleotide and amino acid sequences Tissue expression of catalase Quantification of catalase mRNA expression by real time PCR
Results
Figure 1: The nucleotide and deduced amino acid sequence of catalase from Chinese shrimp F. chinensis. The letters in box indicate the start codon (ATG), the stop codon (TAG) and the polyadenylation signal sequence (AATAAA). The three catalytic amino acids (His-71, Asn-144 and Tyr-354) are shaded by gray and the CAT proximal active site
signature (FDRERIPERVVHAKGAGA) and proximal heme-ligand signature sequence (RLFSYNDTH) are underlined.
Figure 2 : Multiple alignment of the deduced amino acid of the catalase amino acid sequence from F. chinensis with catalase from other species: L. vannamei (AAR99908), A. aegypti (EAT34333), B. mori (NP_001036912), M. musculus (P24270), M. undulates (AAO72713), D. rerio (NP_570987) and H. sapiens (NP_001743). The three conserved catalytic amino acids (His-71, Asn-144 and Tyr-354) are indicated with asterisks and the catalase proximal active site signature (FDRERIPERVVHAKGAGA) and proximal heme-ligand signature sequence (RLFSYNDTH) are framed. Figure 3 : Detection of catalase
transcripts by Real-time PCR. Lanes A, B, C, D, E, F and G for gills, muscle, ovary, intestine, and lymphoid organ, hemocytes respectively. hepatopancreas,
Figure 4 : Analysis of catalase expression in hemocytes of the WSSV challenged group and the control group by Eva Green Real-time PCR at 0, 3, 5, 8, 14, 23, 37 and 59 h post-injection.
Figure 5 : Analysis of catalase expression in hepatopancreas of the WSSV challenged group and the control group by Eva Green Real-time PCR at 0, 3, 5, 8, 14, 23, 37 and 59 h postinjection.
Conclusions 1. A cDNA sequence of catalase gene was cloned from Chinese shrimp F. chinensis. 2. 3. 4. The mRNA encoding catalase was found in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill. The expression of catalase in hemocytes and hepatopancreas changed rapidly and dynamically in response to WSSV infection. The upregulated catalase expression after infection indicated that it was inducible and might be involved in the shrimp immune response. References 1. 2. 3. Iwanaga S, Lee BL. Recent advances in the innate immunity of invertebrate animals. J Biochem Mol Biol. 2005;38(2):128-150. Ken CF, Lin CT, Wu JL, Shaw JF. Cloning and expression of a cDNA coding for catalase from zebrafish (Danio rerio). J Agric Food Chem. 2000 Jun;48(6):2092-2096. Mohankumar K, Ramasamy P. White spot syndrome virus infection decreases the activity of antioxidant enzymes in Fenneropenaeus indicus. Virus Res. 2006;115:69-75.