Generation Of A Fluorescent Transgenic Zebrafish Biosensor For Detection Of Aquatic Heavy Metals

Heavy metal pollution is now one of the hot topics in the study of ecological environment and couldpresent significant harm to development and health of organism and human on account of varioussources, widespreading, uncontrollability and bioaccumulation. Detection and controllingof heavy metalpollution are definitely getting more attention.The experimental virtues of zebrafish including small size, ectogenesis, transparent embryos and cheapmaintenance of the adult have made the zebrafish an excellent and popular vertebrate model. Theavailability of genetic materials and several thousand mutants and transgenic lines together with a broadspectrum of techniques to manipulate gene functions add further to the experimental strength of thismodel in the following fields like vertebrate auxolog，genetics，ecology，basic medicine，drug screeningand toxicology.In order to get recombinant plasmid pEGFP-zMT sensitive to heavy metals,we cloned mt promoter ofzebrafishand inserted itinto eukaryotic expression vector pEGFP-N3with its high active constituteCMV promoter deleted. The present research transferred pEGFP-zMT into one-cell stage zebrafishembryos to achieve transgenic zebrafish expressing GFP via microinjection. Twenty-four hours afterinjection，transgenic zebrafish F0expressing GFP could be examined with fluorescence microscope andbe further verified by polymerase chain reaction and reverse transcription-polymerase chain reaction.The restpositive embryos were collected and maintained. Two specific GFP phenotypes were collectedin F2homozygotes after inbred purification assisted with GFP screening. Type one was pMT-Tü/â… whose epidermal tissues experienced GFP blot and type two was pMT-Tü/â…¡ with intensely extensiveGFP in skin, muscle, bone and other tissues.Aquatic Zn²⁺and Cd²⁺concentrations were designed according to China water quality standard for6h,12h and24h acute toxic assaysusing3dpf,5dpf and10dpf zebrafish embryos as materials in eachtreatment.The result showed that low heavy metal ionic concentrations (1.000mg/L Zn²⁺,2.000mg/LZn²⁺,0.005mg/L Cd²⁺,0.005mg/L Cd²⁺) had no visible effect on development and phenotype ofzebrafish embryos. Developmental deformities including growth inhibition, pericardial cyst andrachiocamposis even deaths were observed in a dosage-dependent manner with the increasing ionicconcentrations. Embryos in different developmental stage showed diverse resistance to the same toxictreatment. Especially,10dpf embryos showed stronger resistance than5dpf embryos and3dpfembryos were weakest.10dpf embryos of zebrafish pMT-Tü/â… and pMT-Tü/â…¡were exposed to Zn²⁺（1.000mg/L,2.000mg/L,5.000mg/L,10.000mg/L）and Cd²⁺（0.005mg/L,0.010mg/L,0.100mg/L）for6h and24h. Real timePCR was designed to measure the quantitative relation between dynamic GFP expression level andheavy metal concentration. The research result indicated that pMT-Tü/â…  and pMT-Tü/â…¡ zebrafishembryos exposed to high concentration Cd²⁺（0.100mg/L） showed higher level of GFP mRNA whileexposure to Zn²⁺strongly inhibited GFP expression in pace with the increasing Zn²⁺concentration for6hours exposure time. Exposure for twenty-four hours showed even more intensely influence. Zn²⁺ treatment definitely inhibited pMT-Tü/â…  embryos GFP expression meanwhile dosage-dependentlyinduced pMT-Tü/â…¡ embryos GFP expression.Cd²⁺treatment for24hours displayed distinctlydosage-dependent GFP mRNA induction to both pMT-Tü/â… and pMT-Tü/â…¡ embryos.The present research finally obtained transgenic zebrafish pMT-Tü/â… and pMT-Tü/â…¡promisinglyto actas biosensor for detection of aquatic heavy metal ions for its inducible expression of report protein GFPand high sensitivity to heavy metal concentrations under China water quality standard.