Construction Of Transgenic Marine Medaka For Monitoring Marine Heavy Metals

Since the Industrial Revolution,marine heavy metal pollution occurred frequently,which has seriously threatened the ecological environment and human health in the coast areas.Biological monitoring of marine heavy metals is an important part of ecological environment protection,and it has attracted the attention of many researchers.The marine medaka（Oryzias melastigma）is small in size,with short sexual maturity time,in vitro development,transparent embryos,and it can be cultured in large numbers in the laboratory.Therefore,such advantages have made it an important model organism in seawater pollution and ecotoxicology research field.In this experiment,we cloned the wild-type marine medaka metallothionein（mt）gene promoter,and modified it to construct eight plasmids containing different modified promoters.Eight plasmids were screened for heavy metal sensitivity through293T cell experiments,and it was found that the p ISce I-Om MT（D1&2）-e GFP plasmid constructed with Om MT（D1&2）as the promoter showed the highest sensitivity to Zn²⁺.At the concentration of 200 u M Zn²⁺,the fluorescence intensity showed the maximum value,and the expression of metallothionein also increases significantly.The expression level of exogenous e GFP demonstrated a positive correlation with the expression level of endogenous mt.Using microinjection technology,the plasmid p ISce I-Om MT（D1&2）-e GFP was transferred to the undivided fertilized eggs of the marine medaka,and the marine medaka Tg（Om MT-e GFP）that could express the green fluorescent gene（e GFP）was selected as the F0 generation.The 1-3 dpf juveniles of Tg（Om MT-e GFP）marine medaka were the used for the exposure experiment.A 72-hour acute exposure test with Cd²⁺,Zn²⁺,Hg²⁺(0,and Cu²⁺.It was found that the transgenic marine medaka is sensitive to heavy metals in the environment(0.005μM Zn²⁺,0.001μM Cd²⁺,0.002μM Cu²⁺and 0.0002μM Hg²⁺),which is lower than the national standard,which indicates that low concentrations of heavy metal cocoa can induce the expression of transgenic marine medaka fluorescent protein.The real-time fluorescence quantitative detection indicate that high concentrations of heavy metals could induce the up-regulation of the expression of Om MT and e GFP genes,and both genes showed a significant positive correlation with the concentrations of heavy metals.Especially in the fish liver,the expression of GFP was significantly enhanced with the increase of the concentration of heavy metals.This study successfully constructed the transgenic marine medaka Tg（Om MT-e GFP）expressed e GFP with highly sensitivity to marine heavy metals——within the range allowed by the national seawater quality standards,and it will be expected to become a real-time and efficient biological sensor for heavy metal detection in the marine environment.At the same time,the research results also provide valuable reference materials for the further use of genetically modified organisms for environmental monitoring research.