Odorant receptor(OR)is crucial for insects to detect and recognize external chemical cues closely related to their survival.The insect OR forms a heteromeric complex composed of a ligand-specific receptor and a ubiqui...Odorant receptor(OR)is crucial for insects to detect and recognize external chemical cues closely related to their survival.The insect OR forms a heteromeric complex composed of a ligand-specific receptor and a ubiquitously odorant receptor coreceptor(Orco).This study used the CRISPR/Cas9 technique to knock out(KO)Orco and reveal its essential role in acting on OR-meditated olfactory behavior in a critical invasive agricultural pest,the fall armyworm(FAW),Spodoptera frugiperda.Electroantennogram(EAG)results suggested that the Orco mutants of both male and female moths severely reduced their electrophysiological responses to the eight tested plant volatiles and two sex pheromones.However,the Orco gene played distinct roles in mating behavior between sexes:the mating behavior was fully disrupted in mutated males but not in mutated females.The oviposition result indicated that the Orco KO females displayed reduced egg laying by 24.1%compared with the mated wild type(WT)females.Overall,these results strongly suggest that Orco is an excellent target for disrupting FAW’s normal behavior and provides a feasible pest control approach.展开更多
ATP-binding cassette transporter C2(ABCC2)is known to be a receptor for Bacillus thuringiensis(Bt)toxins in several lepidopteran insects.Mutations in the ABCC2 gene have been genetically linked to field-evolved resist...ATP-binding cassette transporter C2(ABCC2)is known to be a receptor for Bacillus thuringiensis(Bt)toxins in several lepidopteran insects.Mutations in the ABCC2 gene have been genetically linked to field-evolved resistance to the Cry1 F toxin from Bt in Spodoptera frugiperda.Here we generated a SfABCC2 knockout strain of S.frugiperda using the CRISPR/Cas9 system to provide further functional evidence of the role of this gene in susceptibility and resistance to Cry1 F.Results from bioassays showed that the SfABCC2 knockout S.frugiperda strain displayed 118-fold resistance to Cry1 F compared with the parental DH19 strain,but no resistance to Vip3 A toxin from Bt.These results provide the first reverse genetic evidence for SfABCC2 as a functional receptor for Cry1 F.展开更多
Odorant-binding proteins (OBPs) play key roles in the perception of semiochemicals in insects. Several OBPs in insect olfactory systems have been functionally characterized, and they provide excellent targets for pest...Odorant-binding proteins (OBPs) play key roles in the perception of semiochemicals in insects. Several OBPs in insect olfactory systems have been functionally characterized, and they provide excellent targets for pest control. The functions of some OBPs that are highly expressed in the nonsensory organs of insects remain unclear. Here, the physiological function of an OBP (OBP27) that was highly expressed in the nonsensory organs of Spodoptera frugiperda was studied. OBP27 was nested within the Plus-C cluster according to phylogenetic analysis. The transcription of OBP27 steadily increased throughout the development of S. frugiperda, and transcripts of this gene were abundant in the fat body and male reproductive organs. An OBP27 knockout strain with an early frameshift mutation was obtained using the clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) system. The development time of OBP27^(−/−) larvae was significantly longer than that of other larvae. Both male and female OBP27^(−/−) pupae weighed significantly less than wild-type (WT) pupae. In crosses of OBP27^(−/−) males or females, the mating rate was lower and the mating duration was longer for OBP27^(−/−) male–WT female pairs than for WT–WT pairs. By contrast, the mating rate, hatching rate, and number of eggs of OBP27^(−/−) female–WT male pairs and WT–WT pairs were similar. These findings indicate that OBP27 plays an important role in the larval development and mating process in male adults. Generally, our findings provide new insights into the physiological roles of nonsensory OBPs.展开更多
To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8...To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.展开更多
基金the National Natural Science Foundation of China(32130089)the China Postdoctoral Science Foundation(2020M680785)+3 种基金the Shenzhen Science and Technology Program,China(KQTD20180411143628272)the Central Public-interest Scientific Institution Basal Research Fund of Chinese Academy of Agricultural Sciences(CAAS-ZDRW202108)the Special Funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District,China(PT202101-02)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences(ASTIP).
文摘Odorant receptor(OR)is crucial for insects to detect and recognize external chemical cues closely related to their survival.The insect OR forms a heteromeric complex composed of a ligand-specific receptor and a ubiquitously odorant receptor coreceptor(Orco).This study used the CRISPR/Cas9 technique to knock out(KO)Orco and reveal its essential role in acting on OR-meditated olfactory behavior in a critical invasive agricultural pest,the fall armyworm(FAW),Spodoptera frugiperda.Electroantennogram(EAG)results suggested that the Orco mutants of both male and female moths severely reduced their electrophysiological responses to the eight tested plant volatiles and two sex pheromones.However,the Orco gene played distinct roles in mating behavior between sexes:the mating behavior was fully disrupted in mutated males but not in mutated females.The oviposition result indicated that the Orco KO females displayed reduced egg laying by 24.1%compared with the mated wild type(WT)females.Overall,these results strongly suggest that Orco is an excellent target for disrupting FAW’s normal behavior and provides a feasible pest control approach.
基金supported by the Key Project for Breeding Genetic Modified Organisms of China(2016ZX08012004003)。
文摘ATP-binding cassette transporter C2(ABCC2)is known to be a receptor for Bacillus thuringiensis(Bt)toxins in several lepidopteran insects.Mutations in the ABCC2 gene have been genetically linked to field-evolved resistance to the Cry1 F toxin from Bt in Spodoptera frugiperda.Here we generated a SfABCC2 knockout strain of S.frugiperda using the CRISPR/Cas9 system to provide further functional evidence of the role of this gene in susceptibility and resistance to Cry1 F.Results from bioassays showed that the SfABCC2 knockout S.frugiperda strain displayed 118-fold resistance to Cry1 F compared with the parental DH19 strain,but no resistance to Vip3 A toxin from Bt.These results provide the first reverse genetic evidence for SfABCC2 as a functional receptor for Cry1 F.
基金funded by the National Key R&D Program of China(2021YFD1400700)National Natural Science Foundation of China(31830075).
文摘Odorant-binding proteins (OBPs) play key roles in the perception of semiochemicals in insects. Several OBPs in insect olfactory systems have been functionally characterized, and they provide excellent targets for pest control. The functions of some OBPs that are highly expressed in the nonsensory organs of insects remain unclear. Here, the physiological function of an OBP (OBP27) that was highly expressed in the nonsensory organs of Spodoptera frugiperda was studied. OBP27 was nested within the Plus-C cluster according to phylogenetic analysis. The transcription of OBP27 steadily increased throughout the development of S. frugiperda, and transcripts of this gene were abundant in the fat body and male reproductive organs. An OBP27 knockout strain with an early frameshift mutation was obtained using the clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) system. The development time of OBP27^(−/−) larvae was significantly longer than that of other larvae. Both male and female OBP27^(−/−) pupae weighed significantly less than wild-type (WT) pupae. In crosses of OBP27^(−/−) males or females, the mating rate was lower and the mating duration was longer for OBP27^(−/−) male–WT female pairs than for WT–WT pairs. By contrast, the mating rate, hatching rate, and number of eggs of OBP27^(−/−) female–WT male pairs and WT–WT pairs were similar. These findings indicate that OBP27 plays an important role in the larval development and mating process in male adults. Generally, our findings provide new insights into the physiological roles of nonsensory OBPs.
基金supported by the National Science Foundation of China (30970135)The Key Project of Genetically Modified Organisms Breeding(2009ZX08009-044B)+1 种基金the Natural Science Foundation of Fujian Province of China (No.2006J0065)the Public-interest Scientific Institution Basal Research Fund of Fujian Province (2009R10029-3)
文摘To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.
