Construction Of Bco1 And Bco1l Gene-deficient Zebrafish (Danio Rerio) Model And Assessment Of Toxicity Of Simvastatin To Its Embryo Development

Simvastatin is one of the most widely used statins.Owning to their regulatory functions in lipid metabolism,statins are mainly used in cardiovascular diseases.In consideration of its widespread detection in aquatic environments,the potential adverse effects on aquatic organisms,especially fish,deserve attention.At present,limited information are available on the biotoxicity and bioaccumulation of statins in fish.?-carotene-15,15'-momoxygenase 1(bco1)is a key enzyme in the conversion of ?-carotene to vitamin A metabolism in animals.Vitamin A plays an important role in animal lipid metabolism.bco1 and bco1 l are two major genes encoding ?-carotene-15,15'-momoxygenase 1.The clustered regularly interspaced short palindromic repeats/CRISPR-associated 9(CRISPR/Cas9)system is a novel,third-generation artificial endonuclease that has higher efficiency and less cost compared to other targeted mutation tools.Zebrafish is an outstanding model animal employed in many fields for the advantages of short generation time and large brooding amount and its transparent non-adhesive eggs,which are ideal for microinjection.In the present study,we use CRISPR/Cas9 system to knock out bco1 and bco1 l genes of zebrafish,which encode ?-carotene-15,15'-oxygenase,in order to construct the bco1 and bco1 l gene-deficient zebrafish models for further study of the functions of zebrafish bco1 on vitamin A synthesis and lipid metabolism.The sgRNA recognition sites were selected in the exons region of bco1 and bco1 l genes of zebrafish,and sgRNAs prepared by in vitro transcription were microinjected in the fertilized egg in the first cell stage and the effectiveness was detected.The first fish was constructed by microinjection and screening for heritable mutants.Identification of the basic phenotype of heritable mutants was done by feeding carotene and slicing approaches.In this study,inheritable mutants of bco1 gene and bco1 l gene were obtained,and mutations of bco1 l gene were confirmed to have homozygous lethal effects.The successful establishment of the mutants model of bco1 gene and bco1 l gene provides a reference material for the study of fish carotene metabolism and related developmental process and other follow-up studies.Finally,simvastatin exposure tests were performed on the wild-type and bco1 l mutant zebrafish embryos development.It was found that wild-type zebrafish exposed at a concentration of 5.0 ?g/L for 72 h were abnormal,but the same of performance occured at a concentration of 10 ?g/L for 72 h for bco1 l mutants and showed stronger tolerance.It is required for the next step to explore the possible correlation between bco1 gene,bco1 l gene and fish fat metabolism.