Zebrafish ANO1 Gene Cloning,Eukaryotic Expression And Cellular Function Exploration

Calcium-activated chloride channels?CaCCs?are a family of anion channel proteins widely expressed in a variety of cells and mediate a series of important physiological functions.Using zebrafish fertilized eggs as experimental materials,the CDS region of the zebrafish ANO1?zeb-ANO1?gene was successfully cloned by PCR and analyzed systematically by bioinformatics.The homology analysis was carried out on the ANO1 amino acid sequence of 22 different species,and the phylogenetic tree was established by using the method of N-J.The lentivirus overexpression vector of zeb-ANO1 was constructed and stably transfected into FRT cells,and its overexpression efficiency was detected at the mRNA and protein levels,respectively.The distribution of zeb-ANO1 protein in zebrafish tissues was detected by qPCR and Western blot,and the localization of zeb-ANO1 protein in FRT cells were detected by immunofluorescence assay.The electrophysiological properties of zeb-ANO1 protein were measured by short circuit current experiment,and the effects of classical inhibitors and activators of CaCCs on the activity of zeb-ANO1 were verified.Bioinformatics prediction showed that the amino acid sequence of zeb-ANO1 protein was 925aa with 9 transmembrane structures,which contained several active functional sites,including phosphorylation site,glycosylation site and so on.The transmembrane regions of zeb-ANO1 protein were mostly?-helix conformation and formed spiral barrel,the transmembrane domain of ANO1 protein of different species was conservative,the intracellular domain of N-terminal and C-terminal changed greatly,and the evolution process of ANO1 protein was consistent with the evolution of biological species.The results showed that zeb-ANO1 protein was expressed in the heart,liver,kidney,parotid gland,intestine,spleen and muscle of zebrafish.The expression of zeb-ANO1 was higher in the kidneys,parotid glands,and intestines,which have the function of secreting liquid.The lentivirus over-expression vector of zeb-ANO1 was successfully constructed and stably transfected of Fisher rat thyroid epithelial?FRT?cells were obtained.The zeb-ANO1 protein was mainly localized in the cell membrane and cytoplasm.Its protein activity is activated by Ca²⁺,The effects of CaCC_inh-A01,a classical inhibitor of CaCCs,and ANO1-activator E_actct on zeb-ANO1 activity were similar to those of other CaCCs.In conclusion,the molecular structure and functional expression of zeb-ANO1 protein are similar to those of known ANO1 proteins,so it is believed that the molecular function of zeb-ANO1 protein is also CaCC.In this study,the molecular identification of ANO1 was pushed forward from the primitive amphibians to the more primitive osseous fishes by the first time,which has laid an experimental foundation for the evolutionary study of ANO1protein and its molecular identity and provided a theoretical clue.