[Cloning of full-length cDNA of HMGR from Gobiocypris rarus and analysis of its expression profiles in male exposed to pentachlorophenol].

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the first rate-limiting enzyme in the mevalonate (MVA) pathway. The full-length cDNA of HMGR was cloned from Gobiocypris rarus, and HMGR expression profiles in different tissues and in response to different treatments of pentachlorophenol (PCP) were analyzed by real-time PCR, to investigate the endocrine disruption mechanism of PCP, which altered steroid hormone precursors (cholesterol) levels by modulating gene transcription profiles of HMGR. Based on the homologous clone strategy and rapid-amplification of cDNA ends (RACE) technology, the full-length 3 101-base-pair (bp) cDNA of HMGR was isolated from the livers of rare minnow (Gobiocypris rarus) for the first time, and was designated as GrHMGR (GenBank accession number KF885724). GrHMGR encoded a protein of 884 amino acids and phylogenetic tree analysis indicated that the deduced protein GrHMGR had extensive sequence similarities to other fish HMGRs. Real-time PCR analyses indicated that GrHMGR mRNA expression was tightly controlled in a tissue-specific fashion, with the sites of expression being brain, gonads and liver, and the highest site of expression being gonads. After male rare minnows were exposed to different concentrations of PCP, significant decrease in GrHMGR gene expression with increased PCP concentration in the brain and gonads were observed, together with the differential gene expression trend in the liver. Furthermore, it was found that the decrease of HMGR could reduce the synthesis of cholesterol. This proved that PCP might disrupt the pathway of cholesterol synthesis and then influenced the endocrine system of rare minnow.

[Induction effects of pentachlorophenol on vitellogenin and p53 in Chinese rare minnow (Gobiocypris rarus)].

Taking the Chinese rare minnow (Gobiocypris rarus) as the test animal, the studies were designed to investigate induction effects of pentachlorophenol (PCP) on vitellogenin (VTG) protein, VTG gene and tumor suppressor p53 gene in the liver of Gobiocypris rarus. The endocrine disrupting of PCP was evaluated by detecting VTG, and sensitive biomarkers of PCP were screened at both protein and mRNA levels. Gobiocypris rarus were exposed to PCP at 1.5, 15, 40, 80, 120, 150, 160 microg x L(-1) respectively, while setting blank, solvent control and 17alpha-ethynylestradiol (EE2) as positive control. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), VTG protein expression differences were detected in the liver of Gobiocypris rarus after exposure to PCP. Cloning the VTG and p53 gene new fragments of Gobiocypris rarus based on conserved regions, mRNA expression levels of VTG and p53 gene in the liver of Gobiocypris rarus were determined by quantitative real-time PCR assay after PCP treatment. The results showed that 40, 80, 120, 160 microg x L(-1) PCP induced the liver of male and female Gobiocypris rarus to produce VTG protein, and had a significant concentration effect. VTG and p53 mRNA levels significantly increased in the liver of Gobiocypris rarus after exposure to 1.5, 15, 150 microg x L(-1) PCP, and had remarkable concentration and time effects. The studies suggested that PCP had estrogenic effects, and VTG protein, VTG and p53 gene in the liver of Gobiocypris rarus could be used as candidate sensitive biomarkers for detecting PCP.