| Fish metallothionein(MT) mRNA as a biomarker has been widely used for monitoring heavy metal pollution in aquatic environments, however there were no reports on gender difference from fish MT gene expression under heavy metal exposure. In present study, a detection method for hepatic MT mRNA expression of medaka(Oryzias melastigma), a marine model fish, was optimizaed and established based on real-time quantitative PCR(qPCR) technique. Moreover, the gender difference of hepatic MT mRN A was determined through dose-response and time-course experiments of cadmium with environmental concentrations. Finally it is anticipated that results will play an important role in scientific and technological support for using medaka hepatic MT mRN A as a biomarker to monitor the biological effects of heavy metal in marine environments and will lay an solid foundation to set up and apply a fast, sensitive and specific biomarker for monitoring overall situation of biological effects by heavy metals in marine environment of our country. The achieved major results are shown below.1 The full- length c DNA sequence of MT gene was isolated from O. melastigma for the first time. The full- length cDN A sequence of hepatic MT gene was isolated from medaka by reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of c DNA ends(RACE) technics. The results indicate that hepatic MT cDNA of the medaka contains 385 bp, coding 60 amino acids with a predicted molecular weight 5990 Da and containing the specific sequences of MT gene in vertebrates such as cys-x-cys, cys-x- x-cys and cys- x-cys-cys. The alignment results based on the BLAST searches showed that the amino acid sequence of medaka MT gene exhibited a high identity(82~98%) with other species of piscine.2 The tissues distribution characteristics of MT mRNA in medaka were investigated under normal conditions. The expression profiles of MT mRNA in liver, spleen, intestine, gill, brain and muscle from male and female fish under normal conditions were analyzed by qPCR. The relative expression pattern of MT mRNA from different tissues in both male and female fish is consistent, namely liver > brain > spleen > intestinal > muscle > gill. These results laid the foundation for choosing liver as a appropriate organ to carry on study MT mRN A in response to heavy metal stresses.3 The gender difference of medaka hepatic MT mRNA expression was studied for the first time. After exposure to different concentrations(0.8, 4.0 and 8.0 μg/L) of Cd for 1~7 d, hepatic MT mRN A expressions of all male medaka were almost all significantly increased when compared with the control group, while the hepatic MT mRNA expressions of female medaka were significantly induced only by higher Cd concentrations with longer exposure time, and the induced levels were much lower than that of the male. By comparison of the gender difference of hepatic MT mRNA expression in the present study it is clear that male medaka is much more sensitive than the female, thus it is better to select the male medaka as a sentinel fish. The results indicate that gender difference is an important factor to be considered when using biomarkers for monitoring the biological effects of aquatic pollutants.4 The biological effects of heavy metal pollutants in the scene of para-xylene(PX) explosion accident in a company from Fujian province were successfully monitored by using medaka hepatic MT mRNA expression. In April 2015, explosion accident happened in a PX manufacture company in Fujian province, then we successfully monitored the biological effects of heavy metal pollutants by using the, hepatic MT mRN A of O. melastigma as a biomarker. The results indicate that it is feasible for using medaka hepatic MT mRN A as a biomarker to monitor the biological effects of heavy metal pollution in marine environment. |
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