Cloning And Pcp Impact On DNA Methylation Of Ceruloplasmin Gene In Rare Minnow

Ceruloplasmin,(EC1.16.3Cp) is a kind of important copper transporter, whose role involves in copper transportation, iron metabolism, scavenging of oxyradicals, formation of capillaries and development of the nervous system and embryo. Cp serves as a high efficient, stable and applicable copper source in different stages of development. Besides, Cp is also a marker protein for inflammation, infection, poisoning and cancer. Previous studies in human and mouse yielded substantial data, however very few studies have been conducted in fishes, none for Rare Minnow (Gobiocypris rarus). With the increasing of pollution, environmental monitoring, pollution control and the study of the influence of pollution on the human have been more and more emphasized. As an important part of pollution, water pollution is closely related to the daily life of human beings, which makes it critical for pollution abatement and detection. According to recent study, pentachlorophenol has become the major source of pollution in the river systems of China. Thus it is listed as a priority for monitoring and abatement. Recently, epigenetics has become a hotspot for the research of the impact of low dose pollutants on the damage of organisms.This study is composed of two parts. First, the cloning of the full length cDNA and promoter of Rare Minnow Cp was achieved by RACE and genome walking. Further prediction of protein tertiary structure, construction of phylogenetic tree and distribution of tissue expression were done by bioinformatics analysis. Second, subchronic toxicity test was conducted for the impact of pentachlorophenol on Rare Minnow in the laboratory, according to the levels of actual pollution in the major river systems of China. Accommodative response of Rare Minnow exposed to pentachlorophenol was characterized by the analysis of the difference in methylation of Cp. The main results sre shown as follows:1. The cloning of Cp gene in Rare Minnow was finished by RACE using the cDNA from the liver of Rare Minnow as template. The full length Cp cDNA is3389bp long, with22bp long5’ UTR and103bp long3’UTR,3264bp ORF. It is predicted to encode1087amino acids. Based on phylogenetic analysis, the Cp from Rare Minnow shares the highest homology with the Cp from zebrafish at both nucleotide and amino acid levels for88.1% and90.3%, respectively.2. Based on the5’UTR sequence obtained from RACE, the1430bp promoter region was amplified by genome walking. The core promoter is730bp long. The GC content is40.55%. A full score transcription start site was found in the promoter sequence. Several GATA-1,2,3and CdxA transcription factor binding sites were also found.3. The Cp is shown to have a molecular weight about124429.ID, and pi at6.41. It is predicted lo have nine structural domains such as Al, A2, and B. The secondary structure of the Cp is majorly composed with a-helix,β-sheet, extended strand and random coil, with a percentage at29.53%,7.64%,23.09%and39.74%, respectively. The tertiary structure was shown to be similar to the Cp from zebrafish.4. MEGA5was used to make multiple alignment and construct pbylogenetic tree based on the amino acid sequence of the Rare Minnow Cp. Thirteen species were divided into4groups:mammals, birds and reptiles as a group; amphibian alone as a group; Rare Minnow, zebrafish and channel catfish as a group, four other kinds of fishes as a group.5. Gene expression profile was determined in different tissues by using fluorescence qPCR, β-actin as internal control. Study showed that the Rare Minnow Cp gene was expressed widely in different tissues, with the highest expression level in the livei (P<0.0!). However the expression levels in the rest of tissues were low and the differences were not significant (P>0.05).6. The DNA methylation levels of the Cp gene in liver were determined in Rare Minnow after60days of pentachlorophenol treatment. Study showed that the methylation levels were63.16%(control group),67.54%(0.6μg/L group) and73.68%(6μg/L group) respectively in three different samples. Comparing with the control, the two treated samples were shown to have a4.38%and10.52%increasement in methylation levels, respectively, with significant difference (P<0.01). Between the two treated samples, there is a6.14%increase in methylation levels withsignificant difference (P<0.01). Thus it suggested that the pentachlorophenol treatment was able to increase the methylation levels in the Rare Minnow Cp gene in liver. The rising level of DNA methylation is positively associated with pentachlorophenol concentration.