In mammals,mitofusin 2(MFN2)is involved in mitochondrial fusion,and suppresses the virus-induced RIG-I-like receptor(RLR)signaling pathway.However,little is known about the function of MFN2 in non-mammalian species.In...In mammals,mitofusin 2(MFN2)is involved in mitochondrial fusion,and suppresses the virus-induced RIG-I-like receptor(RLR)signaling pathway.However,little is known about the function of MFN2 in non-mammalian species.In the present study,we cloned an MFN2 ortholog(LcMFN2)in large yellow croaker(Larimichthys crocea).Phylogenetic analysis showed that MFN2 emerged after the divergence of amphioxus and vertebrates.The protein sequences of MFN2 were well conserved from fsh to mammals.LcMFN2 was expressed in all the tissues/organs examined at diferent levels,and its expression was upregulated in response to poly(I:C)stimulation.Overexpression of LcMFN2 inhibited MAVS-induced type I interferon(IFN)promoter activation and antiviral gene expression.In contrast,knockdown of endogenous LcMFN2 enhanced poly(I:C)induced production of type I IFNs.Additionally,LcMFN2 enhanced K48-linked polyubiquitination of MAVS,promoting its degradation.Also,overexpression of LcMFN2 impaired the cellular antiviral response,as evidenced by the increased expression of viral genes and more severe cytopathic efects(CPE)in cells infected with spring viremia of carp virus(SVCV).These results indicated that LcMFN2 inhibited type I IFN response by degrading MAVS,suggesting its negative regulatory role in cellular antiviral response.Therefore,our study sheds a new light on the regulatory mechanisms of the cellular antiviral response in teleosts.展开更多
The large yellow croaker (Larimichthys crocea) is an economically important marine species with the highest annual production among the farmed marine fishes in China. However, the aquaculture industry of this species ...The large yellow croaker (Larimichthys crocea) is an economically important marine species with the highest annual production among the farmed marine fishes in China. However, the aquaculture industry of this species is suffering from severe problems that include weakened disease resistance, decreased growth rate, and reduced meat quality due to frequent inbreeding. Genome editing, which has a huge potential for solving those problems by introducing favorable genetic changes, is not yet available for the large yellow croaker. Here, we pioneered the techniques of embryo microinjection and genome editing using the CRISPR/Cas9 system in this species. Recombinant plasmids encoding green fluorescent protein (GFP) were introduced into the fertilized eggs of L. crocea by microinjection before the chorion had hardened. A high survival rate (40%) and GFP-positive larvae rate (81.8%) were achieved, indicating that the microinjection technique in L. crocea was successfully established. On this basis, Cas9 mRNA and sgRNA targeting the tyrosinase a gene in L. crocea (Lc-tyra) were co-injected into fertilized eggs of L. crocea. Mutant individuals with insertion and deletion mutations of Lc-tyra were detected. These results indicated that the CRISPR/Cas9-based genome editing technology established herein could efficiently introduce mutations at a specific site in the L. crocea genome. This method provides the potential for genetic improvement and functional genomic study in this species. This is the first report on effective CRISPR/Cas9-based genome editing in L. crocea.展开更多
基金This work was supported by National Key Research and Development Program of China under Grant No.2022YFD2401001National Natural Science Foundation of China under Grant No.U1905204+2 种基金China Agriculture Research System of MOF and MARA under Grant No.CARS-47Fujian Science and Technology Department under Grant No.2021N5008Institute of Oceanology of Fuzhou(2021F02).
文摘In mammals,mitofusin 2(MFN2)is involved in mitochondrial fusion,and suppresses the virus-induced RIG-I-like receptor(RLR)signaling pathway.However,little is known about the function of MFN2 in non-mammalian species.In the present study,we cloned an MFN2 ortholog(LcMFN2)in large yellow croaker(Larimichthys crocea).Phylogenetic analysis showed that MFN2 emerged after the divergence of amphioxus and vertebrates.The protein sequences of MFN2 were well conserved from fsh to mammals.LcMFN2 was expressed in all the tissues/organs examined at diferent levels,and its expression was upregulated in response to poly(I:C)stimulation.Overexpression of LcMFN2 inhibited MAVS-induced type I interferon(IFN)promoter activation and antiviral gene expression.In contrast,knockdown of endogenous LcMFN2 enhanced poly(I:C)induced production of type I IFNs.Additionally,LcMFN2 enhanced K48-linked polyubiquitination of MAVS,promoting its degradation.Also,overexpression of LcMFN2 impaired the cellular antiviral response,as evidenced by the increased expression of viral genes and more severe cytopathic efects(CPE)in cells infected with spring viremia of carp virus(SVCV).These results indicated that LcMFN2 inhibited type I IFN response by degrading MAVS,suggesting its negative regulatory role in cellular antiviral response.Therefore,our study sheds a new light on the regulatory mechanisms of the cellular antiviral response in teleosts.
基金The work was supported by grants from the National Key R&D Program of China(2018YFD0900505)National Natural Science Foundation of China(U1905204 and 31802337)+1 种基金China Agricultural Research System(CARS-47)Marine Economic Development Subsidy Fund of Fujian Province(FJHJF-L-2019-2).
文摘The large yellow croaker (Larimichthys crocea) is an economically important marine species with the highest annual production among the farmed marine fishes in China. However, the aquaculture industry of this species is suffering from severe problems that include weakened disease resistance, decreased growth rate, and reduced meat quality due to frequent inbreeding. Genome editing, which has a huge potential for solving those problems by introducing favorable genetic changes, is not yet available for the large yellow croaker. Here, we pioneered the techniques of embryo microinjection and genome editing using the CRISPR/Cas9 system in this species. Recombinant plasmids encoding green fluorescent protein (GFP) were introduced into the fertilized eggs of L. crocea by microinjection before the chorion had hardened. A high survival rate (40%) and GFP-positive larvae rate (81.8%) were achieved, indicating that the microinjection technique in L. crocea was successfully established. On this basis, Cas9 mRNA and sgRNA targeting the tyrosinase a gene in L. crocea (Lc-tyra) were co-injected into fertilized eggs of L. crocea. Mutant individuals with insertion and deletion mutations of Lc-tyra were detected. These results indicated that the CRISPR/Cas9-based genome editing technology established herein could efficiently introduce mutations at a specific site in the L. crocea genome. This method provides the potential for genetic improvement and functional genomic study in this species. This is the first report on effective CRISPR/Cas9-based genome editing in L. crocea.