Omics Analysis Of The Silkworm Silk Gland And Research On The Functions Of Cuticular Proteins In The Silk Gland

Silkworm?Bombyx mori.L?is an important Lepidoptera model insect with a relatively clear genetic background,which is widely studied because of its important biological value and economic value.The exquisite biological structure and the powerful protein synthesis and secretion function of the silk gland has been considered as the basic premise for the silkworm to achieve its important value.However,as of now,the molecular mechanism of specific silk gland structure and protein synthesis and secretion have not been fully revea led,which seriously affected the scope of application of silkworm as a model insect,restrict people to improve the silk properties and to breed high-yielding silkworm strains,and hindered the research process of the silkworm bioreactor and the application progress of silkworm in health,beauty,material etc.This research is mainly about this scientific problem.Focusing on the molecular mechanism of the silk protein synthesis and secretion in the silk gland and the spatial-temporal regulation mechanism,the transcriptomics,proteomics,western,blot,fluorescence quantitative PCR and other molecular biological strategies were employed to perform research on the silk gland,in order to obtain the expression data at the level of transcriptome and proteome,and to provide important theoretical support and perfect data support for the research of silkworm silk gland.The main results in this study are as follows:1.The transcriptome of silk glands were studied systematically by RNA-Seq and other methodsThe transcriptome data of five silk gland parts of the male and female silkworm on the third day of the fifth instar was addressed and deposited in the NCBI?SRA accession number: PRJNA284192?.A total of 7419 genes were expressed in at least one sample,and 5339 and 5592 genes expressed in the anterior silk gland of the female and male silkworm.Compared with other parts of silk gland,282 genes were up-regulated in the ASG.The functional analysis revealed that these genes was mainly involved in the hydrogen ion transport,protein synthesis of cuticular proteins and serine protease inhibitors,and glycolysis.The results suggested that the occurrence of ion transport is an important prerequisite for the conformational change of silk proteins in the silk gland,this process is an energy consuming process driven by ATP,and the glycolysis related proteins in the silk gland provide the necessary ATP for the process.Cuticular proteins were involved in the formation of the endocuticle in the silk gland,while serine protease inhibitors protect silk proteins from degradation during storage in the silk gland.2.The proteome of the different parts of the silkworm silk gland was studied systematically by LC-MS/MS and other techniquesThe proteome data of the anterio r silk gland?ASG?,anterior part of middle silk gland?AMSG?,middle part of middle silk gland?MMSG?,posterior part of middle silk gland?PMSG?and posterior silk gland?PSG?of the silkworm on the third day of the fifth instar was examined,and 1615,2060,2139,2105 and 2132 proteins were detected in the ASG,AMSG,MMSG,PMSG and PSG respectively.Among them,1078 proteins were the common proteins of the five sections of the silk gland,and 364,181,117,138 and 344 proteins were specially high expressed in the five silk gland segments,respectively.By comparative proteome and GO analysis,we found that many specific proteins expressed in the anterior silk gland,which mainly belong to three categories,including cuticular proteins,ion-transport proteins and glycolysis proteins.The results are high consistent with the transcriptome results,implied that these three pathways play an important role in the conformation transition of silk protein and silk fiber formation.3.The classification,distribution and function of the cuticular proteins in the silkworm silk gland were studied by qRT-PCR and other techniquesIn combination with the transcriptome and proteome data of silkworm silk gland,we screened 56 cuticular proteins expressed and up-regulated in the ASG on the third day of the fifth instar.Chromosomal location analysis revealed that 32 cuticular protein genes were distributed in four segments of three chromosomes.The sequences of these four groups of cuticular proteins are highly homologous,which suggested that they were evolved by gene duplication.At the transcriptional level,the expression value of the 42 cuticular proteins in the ASG was more than 100 times higher than in other silk gland sections.At the protein level,19 cuticular proteins were detected only in the anterior silk gland,but not in other sections.By family conservation analysis,the 56 cuticular proteins were classified as several families.There were 31 cuticular proteins in the CPR family,and the other 25 cuticular proteins contain 14 CPH family proteins,5 CPAP family proteins,3 CPG family proteins,2 CPT family proteins and 1 Chitin_bind₃ family proteins.The conserved motifs in the CPH and CPG family are very short,so we carried out the extraction and analysis on conserved motifs of the genes and renamed them.As a result,we newly discovered 8 new cuticular protein families,which laid the foundation for revealing the division and co-evolution of the cuticular proteins from different families.4.The functions of the cuticular proteins with specific sequence motifs in the silk gland of silkworm were studied by western blot and other techniquesWestern Blot was used to determine the expression profiles of four cuticular proteins,including CPR90,CPR68,CPAP3-G and CPAP 3-J,on the third day of the fifth instar silkworm,and from the fourth instar to the fifth instar.These results further confirmed that the CPR family proteins were expressed high in the feeding stage of the fourth and fifth instar,but very low in the molting stage,which is different with the expression pattern of the cuticular proteins in the CPAP family.The CPAP proteins were expressed high in the molt,but decreased rapidly when enter into the feeding stage.The location and dynamic expression pattern in different stages of the cuticular protein CPAP3-G in the anterior silk gland were detected by the immunofluorescence,and the results showed that the CPAP3-G and chitin have the same location in the silk gland,which indicated that CPAP3-G was distributed in the chitin layer.At the same time,the expression pattern of the CPAP3-G in different stages was highly consistent with the Western Blot result.Eight CPAP proteins were found specially expressed in the ASG.We predicted their domains by searching the NCBI website,and then further named them as CPAP3-I,CPAP3-G,CPAP3-H,CPAP1-Q,2 chitinases and 2 chitin deacetylases.According to the results in this study,we deduced the update process of endocuticle layer in the silk gland during the molting stageas follows.CPR cuticular proteins are the main cuticular proteins of the anterior silk gland in the fourth instar,when the chitin layer of the ASG is complete.The chitin layer of the silk gland began to degrade and reconstruct when entering the fourth molting stage.With the rapid decrease of the CPR cuticular proteins,the CPAP-motif cuticular proteins and CPAP-motif chitin degrading enzymes increased sharply,and the chitin would be degraded into the intermediate metabolite,and the CPR proteins were also degraded by proteinases.In the early fifth instar,chitin intermediate metabolites would regenerate into the chitin with the catalysis of a series of enzymes,then the new chitin would combine the new CPR proteins and generate new cuticle layer in the cavity of the anterior silk gland.At the same time,the CPAP proteins accomplished their mission,and would be degraded gradually.Thus,the endocuticle layer of the anterior silk gland completed its renewal process in the molting stage.