title: Molecular cloning, characterization and expression analysis of tumor suppressor protein p53 from orange-spotted grouper, Epinephelus coioides in response to temperature stress.
abstract: The tumor suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. In the present study, the cDNA of p53 from the orange-spotted grouper (Epinephelus coioides) (Ec-p53) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of Ec-p53 was of 1921 bp, including an open reading frame (ORF) of 1143 bp encoding a polypeptide of 380 amino acids with predicted molecular weight of 42.3 kDa and theoretical isoelectric point of 7.0. Quantitative real-time PCR (qRT-PCR) assays revealed that Ec-p53 was ubiquitously expressed in all the examined tissues but with high levels in intestine and liver of the orange-spotted grouper. In addition, we measured the DNA damage and apoptosis in the blood cells and the percentage of dead and damaged blood cells. Our results suggest that oxidative stress and DNA damage occurred in grouper in conditions where the temperature was 15 ± 0.5 °C. Furthermore, qRT-PCR and western blot confirmed that low temperature stress induced upregulation of Ec-p53 in the mRNA and protein levels. These results suggest that low temperature-induced oxidative stress may cause DNA damage or apoptosis, and cooperatively stimulate the expression of Ec-p53, which plays a critical role in immune defense and antioxidant responses.
authors.affiliation: Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, PR China.
authors.forename: Zeng-Hua
authors.initials: ZH
authors.lastname: Qi
authors.forename: Yu-Feng
authors.initials: YF
authors.lastname: Liu
authors.forename: Sheng-Wei
authors.initials: SW
authors.lastname: Luo
authors.forename: Chu-Xian
authors.initials: CX
authors.lastname: Chen
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Wei-Na
authors.initials: WN
authors.lastname: Wang
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D003001:Cloning, Molecular; D020552:Comet Assay / Q000662:veterinary; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D006434:Hemocytes / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010473:Perciformes / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D017422:Sequence Analysis, DNA / Q000662:veterinary; D013312:Stress, Physiological / Q000502:physiology*; D013696:Temperature; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D016159:Tumor Suppressor Protein p53
keywords: DNA damage; Epinephelus coioides; Low temperature stress; Oxidative stress; p53