Study Of Function And Regulation Of Anterior Silk Gland Specific Cuticle Protein1 And 2 In Silkworm,Bombyx Mori

As a representative spinning arthropod,the silkworm（Bombyx mori）is an artificially domesticated economic insect.Silk produced by silkworm is a natural polymeric bio-material.Owing to its good mechanical performance,renewable and degradable properties,silk has been widely used in the traditional textile and clothing industry,medical and health care,beauty,photoelectric conduction,nanomaterials and other emerging industries.Therefore,silk has a broad application prospect in the field of fiber composite materials.Silk proteins are synthesized and secreted by a specialized organ of silkworm,that is silk gland.The liquid silk proteins,including the fibroin secreted by the posterior silk gland（PSG）and the sericin secreted by the middle silk gland（MSG）,flow through the spinning duct and form a solid silk fiber by spinneret.The spinning duct is mainly composed of anterior silk gland（ASG）and spinneret（including its accessory gland,Filippi’s gland）,which is considered as the main place for conformation transformation of silk proteins and silk fibrillogenesis.Previous studies showed that metal ions,p H and shear force are contribute to silk fibrillogenesis.When liquid silk proteins enter the spinning duct,the shear rate of the silk protein increases rapidly as the diameter of the spinning duct decreases sharply.Then silk proteins aggregate,self-assemble under shearing and extensional stresses.The conformation of silk proteins transforms from random-coil and helix conformations to mainlyβ-sheet structures.At the same time,liquid silk becomes liquid crystal state.Furthermore,the silk protein crystals arrange along the fiber axsis under a suitable p H gradient and metal ion strength,and finnaly,the solid fiber is pulled out of the spinneret by head swing.Chitin is the most abundant polysaccharide in nature,which is the main component of exoskeleton and peritrophic matrix of insect.Chitin can combine with different kinds of cuticle proteins and peritrophic matrix proteins to form complex structures for varieties of biological functions in different tissues.Therefore,the interaction between chitin and proteins is crucial for the growth and development of arthropods.As an important place for silk fibrillogenesis,the ASG was analyzed by transcriptomics,proteomics and metabolomics,and a lot of cuticle proteins has been identified.Besides,chitin has been also found in the ASG.However,the biological functions of cuticle proteins and chitin in ASG are still unclear.In this study,we chose the anterior silk gland specific cuticle proteins ASSCP1 and ASSCP2 as targets to investigate the interaction between chitin and cuticle proteins.The mechanism of chitin binding activity for ASSCP1 was studied.The function of the two proteins in the process of silk fibrillogenesis was investigated by means of transgenic RNA interference.Meanwhile,we also explored the tissue-specific regulation of ASSCP2 by transgenic technology.The main achievements and conclusions are as follows:1.Mechanism of chitin binding activity of ASSCP1Bioinformatics analysis revealed that both ASSCP1 and ASSCP2 contain conserved R&R domain and aromatic amino acids.Therefore,we designed site-directed mutation primers based on conserved aromatic amino acid sites.Then,recombinant expression vectors were constructed.The recombinant protein and mutant proteins were obtained by prokaryotic expression and purified by nickel column affinity chromatography.Chitin binding assay were performed.The results showed that the chitin binding abilities of the mutant proteins were weaker than recombinant ASSCP1 protein,indicating that the aromatic amino acids in ASSCP1 were closely related to its chitin binding activity.2.Function of Bm ASSCP1 and Bm ASSCP2 in the process of silk fibrillogenesisWe performed a morphological observation of the ASG and measured the thickness of the cuticular layer during silk spinning.Results showed that the ASG morphology and the thickness of the cuticular layer remained unchanged during the silk spinning process.However,the cuticular layer underwent a dramatic degradation during the pupation stage,with obvious apoptotic characteristics of ASG.Immunofluorescence analysis revealed that chitin and ASSCP1 were co-located in the cuticluar layer.They were constantly presented during silk spinning,and degraded during pupation.To investigate the functions of ASSCP1 and ASSCP2,we successfully obtained two transgenic silkworm lines（ASSCP1-IR and ASSCP2-IR）in which the expression of ASSCP1 and ASSCP2 was knocked down respectively.After knocking down the expression of ASSCP1 and ASSCP2,the thickness of the cuticular layer decreased significantly.Transmission electron microscopy showed that the knocking down disrupted the structural integrity of the cuticular layer and affected the arrangement of chitin.To determine the impacts of knocking down the ASSCP1 and ASSCP2 on the mechanical properties of silk fiber,tensile tests were performed.The results showed that knocking down the ASSCP1 and ASSCP2 has negative impact on mechanical properties of silk.The extensibility and toughness were significantly reduced,indicating that the cuticle proteins and chitin may participate in the silk fibrillogenesis and regulate the mechanical properties of silk.What is more,knocking down the expression of ASSCP1 resulted in smaller silk gland cells and shorter length of ASG.Comparative proteomics analysis of ASSCP1-IR silkworms at the wandering stage showed that the decrease of ASSCP1 affected the expression of other cuticle proteins,suggesting that ASSCP1 may interact with other cuticle proteins and participate in the construction of cuticular layer.3.The study of tissue-specific regulation of Bm ASSCP2ASSCP2 is an anterior silk gland specific cuticle protein of silkworm.Thus,it is of great value to explore the tissue-specific regulation mechanism of ASSCP2.Firstly,the sequentially truncated promoters were cloned.Together with the reporter gene EGFP,the sequentially truncated promoters were inserted into piggy Bac vector.After microinjection,we successfully obtained the transgenic positive silkworm lines,named as Bm CP2P（full length）,Bm CP2P^T1108,Bm CP2P^T757,Bm CP2P^T448,Bm CP2P^T357,Bm CP2P^T257and Bm CP2P^T157.To investigate the expression of repoter gene EGFP,the fluorescence observation and Western blot were carried out.The results showed that Bm CP2P can specifically directed heterologous protein expression in the ASG until the promoter was truncated to 357 bp.When truncated to 257 bp and 157 bp,the promoter lost its activity.This implied that there are transcription activation factors binding sites in the-357～-257 bp upstream of ASSCP2 gene.EMSA showed that there were tissue-specific transcription factors in the anterior silk gland that could specifically bind to the-357～-257 bp probe.We also screened the silkworm genome database and identified a transcriptional factor Bm Sox2,which is expressed in ASG specifically.We found that recombinant Bm Sox2 could also specifically bind to the-357～-257bp probe,indicating that it might be involved in the tissue-specific expression regulation of ASSCP2.In summary,this study investigated the functions of ASSCP1,ASSCP2 and chitin in the spinning duct.We found that the cuticle protein is co-located and co-expressed with chitin in the cuticular layer of the spinning duct.The cuticle proteins can interact with chitin,form the hard and rigid cuticular layer for supporting the cells and participate in silk fibrillogenesis and spinning,regulate the mechanical properties of silk.Thus,the cuticle proteins may be a potential target for the improvement of the mechanical properties of silk.Besides,the mechanism of tissue-specific regulation of ASSCP2 gene was investigated,and it was found that-357～-257bp sequence was involved in transcriptional activation of ASSCP2 gene.An ASG specific transcription factor Bm Sox2 was identified,which can be a reference for the study of specific expression mechanism in the ASG.