Few studies have reported obtaining grass carp resistant to hemorrhagic disease via gene editing in commercial fish. Here, we demonstrate that the expression and activity of grass carp PI4KB (gcPI4KB) are vital for GC...Few studies have reported obtaining grass carp resistant to hemorrhagic disease via gene editing in commercial fish. Here, we demonstrate that the expression and activity of grass carp PI4KB (gcPI4KB) are vital for GCRV-I and GCRV-II replication. Given the obvious cytopathic effect (CPE) in the present available cell lines is only caused by GCRV-I, but GCRV-II is the current popular and fatal strain in grass carp, GCRV-I and GCRV-II are used in cell lines and in grass carp, respectively. In vitro studies in CIK cells revealed that gcPI4KB interacted with NS80 and VP3 of GCRV-I, and that gcPI4KB was recruited by NS80 for promoting the generation of GCRV viral inclusion bodies (VIBs). Since the negative regulatory role of gcPI4KB in GCRV infection was confirmed by in vitro data, we performed gene editing of gcPI4KB in grass carp. We found that PI4KB F0 juvenile grass carp crispants have obvious advantages in promoting growth and in resisting GCRV-II infection. Compared with uninfected WT grass carp, the uninfected PI4KB F0 juvenile grass carp crispants exhibit a higher expression level of many genes involved in growth- and development-related metabolic pathways such as the FoxO signaling pathway and insulin signaling pathway. Compared with WT grass carp without infection, PI4KB F0 juvenile grass carp crispants without infection or WT grass carp infected with GCRV-II, higher expression levels for many genes involved in metabolic diseases and viral infections were observed in the liver from PI4KB F0 juvenile grass carp crispants infected with GCRV-II. Altogether, the present study suggests the mechanism of gcPI4KB in facilitating GCRV replication, the signaling pathways regulated by gcPI4KB, and the possibility to obtain grass carp resistant to hemorrhagic disease via gene editing of PI4KB.展开更多
Fishes exposed to different dissolved oxygen concentrations over prolonged time periods have different susceptibility and adaptation mechanisms.In this study,healthy grass carp with an average weight of 214.6±18....Fishes exposed to different dissolved oxygen concentrations over prolonged time periods have different susceptibility and adaptation mechanisms.In this study,healthy grass carp with an average weight of 214.6±18.7 g were selected for long-term culture in recirculating aquaculture systems at three different dissolved oxygen concentrations,i.e.,3.00 mg/L[(2.57±0.33)mg/L,DO_(2.57)),5.00 mg/L[(4.61±0.11)mg/L,DO_(4.61)],which was the control group,and 7.00 mg/L[(6.50±0.48)mg/L,DO_(6.50)].Blood,gill,muscle,and liver samples were collected after 60 days.Using hematoxylin-eosin(HE),alcian blue periodic acid Schiff(AB-PAS)staining,determination of respiratory metabolism enzyme activity,and fluorescent quantitative PCR,the adaptability of juvenile grass carp facing different dissolved oxygen levels were explored.The results showed that the respiration rate increased,and hemoconcentration and hemoglobin(Hb),electrolyte and pH changed at DO2.57.Furthermore,gill lamellae became thinner and longer,the distance between them increased,the mucus on them decreased and the interstitial cell mass decreased.In addition,the relative expression of apoptosis-related genes bcl-2 and bcl-xl in gill tissue decreased,while caspase-3 and bax increased.In muscles and liver tissues,lactate dehydrogenase(LDH)activity and lactic acid content(LA)increased,pyruvate dehydrogenase(PDH)activity decreased,and genes related to the respiratory metabolic enzymes PDH-A4 and PDH-E1α also changed accordingly.The oxygen consumption rate decreased significantly(P<0.05).At DO_(6.50),the distance between gill lamellae,numbers of red blood cells(RBC)and the content of hemoglobin decreased wheras the mucus on gill lamellae increased.The activity of PDH,the expression of PDH-E1a in liver and oxygen consumption rate increased.Different dissolved oxygen concentrations affected the respiratory behavior,gill tissue structure,blood transport capacity and tissue enzyme activity of juvenile grass carp.In summary,juvenile grass carp showed different adaptation strategies when facing different dissolved oxygen concentrations over prolonged periods in recirculating aquaculture systems.展开更多
基金supported by Biological Breeding-Major Projects(2023ZD04065)the National Key Research and Development Program(2023YFD2401603)National Natural Science Foundation of China(U22A20535 and 32373180).
文摘Few studies have reported obtaining grass carp resistant to hemorrhagic disease via gene editing in commercial fish. Here, we demonstrate that the expression and activity of grass carp PI4KB (gcPI4KB) are vital for GCRV-I and GCRV-II replication. Given the obvious cytopathic effect (CPE) in the present available cell lines is only caused by GCRV-I, but GCRV-II is the current popular and fatal strain in grass carp, GCRV-I and GCRV-II are used in cell lines and in grass carp, respectively. In vitro studies in CIK cells revealed that gcPI4KB interacted with NS80 and VP3 of GCRV-I, and that gcPI4KB was recruited by NS80 for promoting the generation of GCRV viral inclusion bodies (VIBs). Since the negative regulatory role of gcPI4KB in GCRV infection was confirmed by in vitro data, we performed gene editing of gcPI4KB in grass carp. We found that PI4KB F0 juvenile grass carp crispants have obvious advantages in promoting growth and in resisting GCRV-II infection. Compared with uninfected WT grass carp, the uninfected PI4KB F0 juvenile grass carp crispants exhibit a higher expression level of many genes involved in growth- and development-related metabolic pathways such as the FoxO signaling pathway and insulin signaling pathway. Compared with WT grass carp without infection, PI4KB F0 juvenile grass carp crispants without infection or WT grass carp infected with GCRV-II, higher expression levels for many genes involved in metabolic diseases and viral infections were observed in the liver from PI4KB F0 juvenile grass carp crispants infected with GCRV-II. Altogether, the present study suggests the mechanism of gcPI4KB in facilitating GCRV replication, the signaling pathways regulated by gcPI4KB, and the possibility to obtain grass carp resistant to hemorrhagic disease via gene editing of PI4KB.
基金supported by the earmarked fund for CARS(CARS-45)the Fundamental Research Funds for the Central Universities(No.2662022SCYJ001)the National Key R&D Program of China(No.2019YFD0900303).
文摘Fishes exposed to different dissolved oxygen concentrations over prolonged time periods have different susceptibility and adaptation mechanisms.In this study,healthy grass carp with an average weight of 214.6±18.7 g were selected for long-term culture in recirculating aquaculture systems at three different dissolved oxygen concentrations,i.e.,3.00 mg/L[(2.57±0.33)mg/L,DO_(2.57)),5.00 mg/L[(4.61±0.11)mg/L,DO_(4.61)],which was the control group,and 7.00 mg/L[(6.50±0.48)mg/L,DO_(6.50)].Blood,gill,muscle,and liver samples were collected after 60 days.Using hematoxylin-eosin(HE),alcian blue periodic acid Schiff(AB-PAS)staining,determination of respiratory metabolism enzyme activity,and fluorescent quantitative PCR,the adaptability of juvenile grass carp facing different dissolved oxygen levels were explored.The results showed that the respiration rate increased,and hemoconcentration and hemoglobin(Hb),electrolyte and pH changed at DO2.57.Furthermore,gill lamellae became thinner and longer,the distance between them increased,the mucus on them decreased and the interstitial cell mass decreased.In addition,the relative expression of apoptosis-related genes bcl-2 and bcl-xl in gill tissue decreased,while caspase-3 and bax increased.In muscles and liver tissues,lactate dehydrogenase(LDH)activity and lactic acid content(LA)increased,pyruvate dehydrogenase(PDH)activity decreased,and genes related to the respiratory metabolic enzymes PDH-A4 and PDH-E1α also changed accordingly.The oxygen consumption rate decreased significantly(P<0.05).At DO_(6.50),the distance between gill lamellae,numbers of red blood cells(RBC)and the content of hemoglobin decreased wheras the mucus on gill lamellae increased.The activity of PDH,the expression of PDH-E1a in liver and oxygen consumption rate increased.Different dissolved oxygen concentrations affected the respiratory behavior,gill tissue structure,blood transport capacity and tissue enzyme activity of juvenile grass carp.In summary,juvenile grass carp showed different adaptation strategies when facing different dissolved oxygen concentrations over prolonged periods in recirculating aquaculture systems.
