Generation And Application Of A Transgenic Marine Medaka (Oryzias Melastigma)

Dioxin-like compounds(DLCs)are persistent organic pollutants.DLCs are able to bind aryl hydrocarbon receptor(AhR)and produce the toxic effect.The level of cytochrome P450 1A1(CYP1A1)is a typical biomarker indicating the activation of AhR signaling.With the development of industry and human life,a variety of DLCs are produced and enter the world chemical cycle.DLCs widely distribute in the ocean.In this study,the cyp1a1 promoters from different species and the zebrafish cyp1a1 promoters containing the different number of dioxin response element(DRE)were compared by molecular cloning techniques.The sensitive recombinant luciferase plasmid was selected for building the recombinant cell line.The recombinant cell line was applied in screening AhR agonists.The sensitivity of the zebrafish cyp1a1-12DRE promoter is higher than the marine medaka cyp1a1 promoter and the recombinant mice cyp1a1 promoter in previous study.The Tg(cyp1a-12DRE:egfp)transgenic marine medaka was generated and applied in detecting AhR agonists in the seawater.The transgenic marine medaka possessed high sensitivity and specificity as well as low EGFP background.The high concentrations of AhR agonists in the seawater samples from Quangang seaside successfully detected by Tg(cyp1a-12DRE:egfp)larvae after pollution accident.The method of seawater enrichment then dilution is suitable for detecting seawater which is the concentration lower than the sensitivity of transgenic marine medaka.It effectively increased the detected limit in this method.The concentrations of AhR agonists in the seawater extracts from Dalian Bay and Jinzhou Bay detected by Tg(cyp1a-12DRE:egfp)larvae and GC-MS analyses.The correlation was high between these two methods.This result indicated the method of transgenic marine medaka possess high accuracy.Behavioural evaluations indicated that the increasing of anxiety-like response,predator avoidance and the decreasing of aggression,shoaling,learning and memory ability in marine medaka long-term exposed to environmental-realistic levels of diuron.The reproductive behaviour was also disorder.The reason why some behaviours disorder is that the dopamine level was decreased in the brain of marine medaka.The gonado somatic index and the motility of sperm was significantly decreased in the male marine medaka exposed to environmental-realistic levels of diuron for 180 days.The development and mature of testis was significantly inhibited.The relative mRNA expression of metabolizing enzyme cyplal,cyp1b1,ugt1,ugt2 was significantly increased and activated by AhR signaling in the liver.A great deal of diuron was transferred to DCPMU and DCPU catalyzed by metabolizing enzyme.The mRNA transcription of hypothalamus-pituitary-gonadal-liver-axis was disorder.GnRH,FSH and LH was significantly increased.The survival rate in F1 embryos of male was significantly decreased.The malformation rate of F1 larvae was significantly increased.The gonado somatic index was significantly decreased in the female marine medaka exposed to environmental-realistic levels of diuron for 180 days.The development and mature of ovary was significantly inhibited.The relative mRNA expression of metabolizing enzyme cyp1a1,cyp1b1,ugt1,sult1 was significantly increased and activated by AhR signaling in the liver.A great deal of diuron was transferred to DCPMU and DCPU catalyzed by metabolizing enzyme.The mRNA transcription of hypothalamus-pituitary-gonadal-liver-axis was disorder.GnRH,FSH,E2 and E2/T was significantly decreased.The malformation rate in F1 larvae of female was significantly increased.The effective method based on the transgenic marine medaka is sensitive,specific,cheap,fast,and convenient.It provides a new idea for evaluating ecological toxicity risk of DLCs pollution in the sea.This study reveals the mechanism that metabolizing enzyme activated by AhR signaling in the marine medaka exposed to diuron and the toxicity caused by diuron metabolite.It helps to understand diuron and other AhR agonist pesticides has a long-term effect on fitness of marine fish.It is of great significance for the protection of marine environment and fish resources.