Cloning,Expression and Immune Function Analysis of MAVS Gene in Carp
[Objective]This study was aimed to obtain the CDS region of mitochondrial antiviral signaling protein(MAVS)gene in carp,perform bioinformatics analysis on it,and explore the function of MAVS gene in immune response.[Method]Referring to the predicted sequence of MAVS gene in Cyprinus carpio published in NCBI(GenBank accession No.:XM_019072216.2),primers were designed for its cloning and sequencing,the property and function of MAVS protein in carp were analyzed using bioinformatics softwares.Carp was subjected to Aeromonas hydrophila challenge experiment,and Real-time quantitative PCR was used to detect the expression of MAVS gene in various tissues of carp before and after challenge.Experimental subcellular localization of MAVS and mitochondria was performed by immunofluorescence assay.The expression of MAVS-regulated downstream genes was detected using dual-luciferase reporter gene system.[Result]The CDS region of MAVS gene in carp was cloned to a length of 1 749 bp,encoding a total of 582 amino acids.The similarities of the nucleotide sequences of MAVS gene in carp with those of Carassius auratus,Onychostoma macrolepis,Puntigrus tetrazona,Labeo rohita,Ctenopharyngodon idella,Megalobrama amblycephala,Rhinichthys klamathensis goyatoka,Sinocyclocheilus anshuiensis and Pimephales promelas were 92.3％,89.5％,85.1％,79.1％,73.6％,73.1％,72.0％,70.1％and 69.8％,respectively.The results of phylogenetic tree showed that carp clustered into a single unit with Carassius auratus,Labeo rohita,Puntigrus tetrazona,etc.MAVS protein of carp was an acidic,unstable protein with strong hydrophilicity,containing 133 phosphorylation sites and 1 transmembrane helix,which belonged to transmembrane proteins.MAVS protein did not contain a signal peptide,and did not belong to secretory proteins.Protein conservation analysis results showed that the N-terminal CARD structural domain,the middle proline-rich structural domain(PRR)and the C-terminal transmembrane structural domain(TM)of MAVS were highly conserved among different species.The secondary structure and tertiary structure of MAVS protein in carp showed that the protein was mainly composed of random coil(58.93％)and alpha helix(23.37％),with low contents of extended chain(13.92％)and beta turn(3.78％),suggesting that it was a hybrid protein.Protein-protein interaction results showed that there were interactions between MAVS protein and immune proteins such as NLRP3,TRAF6,ATG5,DDX58 and TBK1.Real-time quantitative PCR results showed that compared with control group,the expression of MAVS gene in heart,brain,spleen,liver,intestines,muscle and gill of carp were significantly or extremely significantly increased after challenge(P＜0.05 or P＜0.01),the expression in kidney was extremely significantly decreased(P＜0.01),and there was no significant difference in eye tissues(P＞0.05).Cellular immunofluorescence results showed that the expression location of MAVS protein was consistent with mitochondria.The results of dual luciferase gene assay reported that MAVS gene could extremely significantly activate IFN and ISRE promoters(P＜0.01).[Conclusion]MAVS gene was highly conserved across species and during genetic evolution,and was widely expressed in different tissues of carp.The results provided a theoretical basis for further study on the function and molecular mechanism of MAVS gene in innate immune response of carp.
万方数据
维普
