Chitin is an abundant natural biopolymer that plays a crucial role in insect growth and development as a fundamental structural component of the exoskeleton.The membrane-integralβ-glycosyltransferase,chitin synthase,...Chitin is an abundant natural biopolymer that plays a crucial role in insect growth and development as a fundamental structural component of the exoskeleton.The membrane-integralβ-glycosyltransferase,chitin synthase,has been identified as the central component in chitin biosynthesis.However,the precise roles of other proteins in facilitating chitin synthase in chitin biosynthesis remain unclear.In this study,we employed split-ubiquitin membrane yeast two-hybrid(MYTH)and pull-down assays to demonstrate the physical interaction between Twinstar(Tsr),a small molecular protein in the actin-depolymerizing factor ADF/Cofilin protein family,and chitin synthase Krotzkopf verkehrt(Kkv)in Drosophila melanogaster in vitro.The RNA interference(RNAi)-mediated global knockdown of Tsr in D.melanogaster resulted in larval lethality.Furthermore,targeted suppression of Tsr in the tracheal and epidermal tissues also led to larval mortality,while knocking down Tsr in the wing tissues led to wrinkled wings.Additionally,silencing Tsr not only reduced the chitin content in the first longitudinal vein of the wings but also led to the absence of the chitin lamellar structure.To validate the functional conservation of Tsr in other insect orders,the two agricultural pests Ostrinia furnacalis and Tribolium castaneum,representing lepidoptera and coleoptera insects,respectively,were investigated.Knockdown experiments targeting the Drosophila Tsr orthologues OfTsr in O.furnacalis and TcTsr in T.castaneum produced abnormal larvae during molting or pupation in O.furnacalis and lethality in T.castaneum.Our findings not only improve ourknowledge of the chitin biosynthesis machinery in insect cuticles but also provide new potential targets for the control of major agricultural pests.展开更多
The insect cuticle,which serves as both a protective barrier and an efficient lever system for locomotion,is an extracellular matrix primarily composed of chitin and protein.The cuticle protein CPCFC characterized by ...The insect cuticle,which serves as both a protective barrier and an efficient lever system for locomotion,is an extracellular matrix primarily composed of chitin and protein.The cuticle protein CPCFC characterized by a“CFC”motif containing 2 Cys split by the insertion of 5 residues is distributed across most insect species and specifically localized in the hard part of the cuticle.However,their physiological function is not fully understood.Here,we report 2 CPCFC proteins,TcCPCFC1 and TcCPCFC2,derived from the Coleopteran insect Tribolium castaneum.We revealed that TcCPCFC1 and TcCPCFC2 were predominantly expressed during the larval and adult stages of T.castaneum,respectively.The transcription downregulation of TcCPCFC1 significantly decreased the modulus and toughness of the elytral cuticle.We found that TcCPCFC proteins have high binding affinity to chitin.We cloned and produced recombinant TcCPCFC proteins and demonstrated that the addition of TcCPCFC proteins to chitin hydrogel greatly enhanced the hydrogel's modulus and toughness by forming denser chitin fibrous networks.Our findings reveal the functional role of CPCFC proteins in enhancing mechanical properties of insect cuticle,and we validate this process in vitro,and offer a protein candidate for fabrication of advanced chitin-based materials.展开更多
基金supported by the National Natural Science Foundation of China(32161133010)the National Key Research and Development Program of China(2022YFD1700200)+2 种基金the Innovation Program of Chinese Academy of Agricultural Sciences(CAAS-CSCB-202302)the Shenzhen Science and Technology Program,China(KQTD20180411143628272)the Special Funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District,China(PT202101-02).
文摘Chitin is an abundant natural biopolymer that plays a crucial role in insect growth and development as a fundamental structural component of the exoskeleton.The membrane-integralβ-glycosyltransferase,chitin synthase,has been identified as the central component in chitin biosynthesis.However,the precise roles of other proteins in facilitating chitin synthase in chitin biosynthesis remain unclear.In this study,we employed split-ubiquitin membrane yeast two-hybrid(MYTH)and pull-down assays to demonstrate the physical interaction between Twinstar(Tsr),a small molecular protein in the actin-depolymerizing factor ADF/Cofilin protein family,and chitin synthase Krotzkopf verkehrt(Kkv)in Drosophila melanogaster in vitro.The RNA interference(RNAi)-mediated global knockdown of Tsr in D.melanogaster resulted in larval lethality.Furthermore,targeted suppression of Tsr in the tracheal and epidermal tissues also led to larval mortality,while knocking down Tsr in the wing tissues led to wrinkled wings.Additionally,silencing Tsr not only reduced the chitin content in the first longitudinal vein of the wings but also led to the absence of the chitin lamellar structure.To validate the functional conservation of Tsr in other insect orders,the two agricultural pests Ostrinia furnacalis and Tribolium castaneum,representing lepidoptera and coleoptera insects,respectively,were investigated.Knockdown experiments targeting the Drosophila Tsr orthologues OfTsr in O.furnacalis and TcTsr in T.castaneum produced abnormal larvae during molting or pupation in O.furnacalis and lethality in T.castaneum.Our findings not only improve ourknowledge of the chitin biosynthesis machinery in insect cuticles but also provide new potential targets for the control of major agricultural pests.
基金supported by the Shenzhen Science and Technology Program(Grant No.KQTD20180411143628272)the National Natural Science Foundation of China(32161133010 and 32200393)+1 种基金the National Key Research and Development Program of China(2022YFD1700200)the Special Funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District(PT202101-02).
文摘The insect cuticle,which serves as both a protective barrier and an efficient lever system for locomotion,is an extracellular matrix primarily composed of chitin and protein.The cuticle protein CPCFC characterized by a“CFC”motif containing 2 Cys split by the insertion of 5 residues is distributed across most insect species and specifically localized in the hard part of the cuticle.However,their physiological function is not fully understood.Here,we report 2 CPCFC proteins,TcCPCFC1 and TcCPCFC2,derived from the Coleopteran insect Tribolium castaneum.We revealed that TcCPCFC1 and TcCPCFC2 were predominantly expressed during the larval and adult stages of T.castaneum,respectively.The transcription downregulation of TcCPCFC1 significantly decreased the modulus and toughness of the elytral cuticle.We found that TcCPCFC proteins have high binding affinity to chitin.We cloned and produced recombinant TcCPCFC proteins and demonstrated that the addition of TcCPCFC proteins to chitin hydrogel greatly enhanced the hydrogel's modulus and toughness by forming denser chitin fibrous networks.Our findings reveal the functional role of CPCFC proteins in enhancing mechanical properties of insect cuticle,and we validate this process in vitro,and offer a protein candidate for fabrication of advanced chitin-based materials.
