Indentification And Study Of Two Novel Cholinesterase-like Genes In Zebrafish

Cholinesterase is a kind of glycoprotein in the form of isoenzyme.It mainly includes Acetylcholinesterase(AChE)and Butylcholinesterase(BChE).AChE can hydrolyze the neurotransmitter Acetylcholine(ACh),but the biological function of BChE has not been fully elucidated.Zebrafish is an important vertebrate model.Studies have identified AChE gene of zebrafish.Although zebrafish has BChE activity,its BChE homologous gene has not been identified yet.Zebrafish unnamed genes si:dkey-30c15.1 7,si:ch73-89b15.3 have been predicted to have cholinesterase domains in the database,but have not been identified.Therefore,the study on the cholinesterase activity of these two genes can provide important theoretical basis and experimental basis for the identification of the genes encoding BChE active protein in zebrafish and the study on their functions,as well as the study on the function of the whole cholinesterase gene family.ObjectiveTo analyze the expression patterns of gene si:dkey-30c15.17 and si:ch73-89b15.3 in zebrafish and explore if they had cholinesterase activity.Contents1.BChE activity verification in zebrafishCollected embryos after fertilization(0,1,2,3,4,5,6 dpf)of zebrafish and samples of adult zebrafish tissues(intestine,liver,brain,testis,ovary,heart,kidney),and verified the activity of BChE.2.Expression patterns of two cholinesterase-like genes in zebrafish The expression and distribution of two zebrafish cholinesterase-like genes si:dkey-30c15.1 7 and si:ch73-89b15.3 in the early embryonic development of zebrafish and in the adult tissues were studied by RT-PCR.3.Overexpression of two cholinesterase-like genes in HEK293T cells and detection of their cholinesterase activity.The expression plasmids of two zebrafish cholinesterase-like genes in eukaryotic cells were constructed and transfected into HEK293T cells to detect the cholinesterase activity of overexpressed cells.4.Two cholinesterase-like genes knockout homozygotes constructed byCRISPR/Cas9 technology and its cholinesterase activity were detected.CRISPR/Cas9 mRNA target sites were designed for two cholinesterase-like genes si:dkey-30c15.17 and si:ch73-89b15.After preparing gRNA and Cas9 mRNA,injecting mixture into zebrafish embryos,serial subcultivation and selection,we got two cholinesterase-like genes knockout homozygotes.Finally,detected its cholinesterase activity.Results1.High BChE activity was detected in the intestine,liver,brain of adult zebrafish.In the zebrafish embryos of 5 dpf,obvious BChE activity was detected,which confirmed the presence of BChE activity in zebrafish.2.In adult zebrafish tissues,si:dkey-30c15.17 and si:ch73-89b15.3 were mainly expressed in the liver,gonad,kidney and brain tissues of zebrafish.In zebrafish embryos,si:dkey-30c15.1 7 and si:ch73-89b15.3 had high expression levels at 2,5,6 dpf and 5,6 dpf,respectively.3.Two zebrafish cholinesterase-like genes si:dkey-30c15.17,si:ch73-89b15.3 was overexpressed in HEK293T cells,and no BChE activity or AChE activity was detected in the overexpressed cells.4.The homozygous mutants of zebrafish genes si:dkey-30c15.17 and si:ch73-89b15.3 were successfully constructed by using the CRISPR/Cas9 technology.In the tissues of adult zebrafish mutants,the cholinesterase activity was not significantly different from that of the wild type,suggesting that si:dkey-30c15.17 and si:ch73-89b15.3 genes had neither BChE activity nor AChE activity.