| Insects are the most diverse animal population in nature,and many insects secrete biological adhesives to protect themselves,nests,or their eggs.Biological adhesives secreted by insects have good adhesion properties and are often able to maintain long-term stickiness in wet,dirty environments.As a representative insect of the order Lepidoptera,silkworm has multiple tissues and organs that secrete substances with viscous effects,such as silk glands and colleterial glands.The colleterial gland is an accessory organ of the female reproductive system of silkworm.It develops slowly in the early stage of pupae,but develops rapidly before pupae emergence.It also produces a large amount of mucus,which is coated on the back of the egg during egg laying and used to anchor the egg on mulberry leaf or silkworm seed paper.Many studies have focused on the morphology of insect colleterial glands and the physicochemical properties of mucus,and it has been found that the main component of mucus is egg glue protein.In addition,it was found that these EGP are generally rich in amino acids such as glycine proline and serine.However,the sequence of EGP secreted by insect colleterial glands has not been reported yet.Therefore,it is still unknown what structure gives the excellent viscosity of EGP.This study focused on these questions in depth.This study on the mucous glands of female silkworm moth transcriptome sequencing,screening to express a high percentage of genes in colleterial glands,LC-MS/MS was used to the most obvious stripe in the mucous mass spectrometry,a candidate of eggs glue proteins were identified,obtained full of eggs through 5' and 3' RACE glue protein-coding genes,and the sequence structure analysis glycosylation and adhesive performance research to obtain the main results are as follows:1.Identification and expression analysis of EGP in silkwormThe vastal surface of silkworm eggs was observed by stereomicrograph and electron microscopy.It was found that there was a transparent gelatinous film on the vastal surface of silkworm eggs,which was formed after the drying of the viscous substance secreted by colleterial glands.In order to identify the proteins that play sticky functions,we used urea to elute the egg surface and performed SDS-PAGE.The results showed that the eluted protein content of the egg surface was relatively rich,and the main band was located at 280 k Da.The same gene was identified through the glial spectrum identification of the main band digging and transcriptome analysis of the colleterial glands of the female adult silkworm,and the gene sequence was retrieved in the Silk Base database to identify an unknown functional protein,but the gene sequence was not complete.Therefore,we used the 5'RACE method to obtain the complete gene sequence,which we named egg glue protein.The 23 amino acids encoded by the first exon,Md PKHYGVLAVLLLSTFIFWVDG,were predicted to be the signal peptide of the egg glue protein.Exon 19 is a short sequence of highly repetitive region,consisting of 5 amino acids as a repeating unit,which was repeated 346 times in total.The most repeated unit is GGNQQ,among which Gly at position 1 and 2 is very conservative,and Asn at position3 and Gln at position 4 are sometimes replaced by acidic residues Glu and Asp or alkaline residues Lys.Pro occasionally replaces Gln at number 5.There are two cysteines in exon21.By electrophoresis,it is found that the egg glue protein can form disulfide bonds.It was found that the expression pattern of egg glue protein was only high in colleterial glands of female adults.Further study showed that egg glue protein was mainly synthesized in the secretory part of colleterial glands.The tissue expression pattern of female from pupal stage to moth stage was analyzed,and it was found that the expression level of egg glue protein started from the third day of pupa,gradually increased with the development of pupa,reached the highest value on the day of emergence of female moth,and gradually decreased after emergence.Immunofluorescence localization was used to observe the distribution of egg glue protein in the storage and secretory parts of colleterial glands.It was found that egg glue protein was synthesized from the dendritic secretory parts and secreted into the lumen,and then flowed to the enlarged lumen of the storage department for storage.2.Prokaryotic expression,Eukaryotic Expression,Purification and Structural Analysis of EGP in SilkwormIt was found that the sequence structure was mainly composed of signal peptide,Nterminal,repeat region and C-terminal.The repeat region was mainly composed of highly repeated 5 peptide GGNQQ with 346 repeats.The repeat region was characterized by high repeatability and small repeat unit.To explore the repeat area influence on egg protein structure and function,through the method of eukaryotic cells express and expressed in prokaryotic cells to repeat sequence capture segments by protein expression,expressed in prokaryotic cells and eukaryotic cells expressing the restructuring of the egg glue protein by nickel column affinity chromatography and gel filtration chromatography purification,get a lot of pure restructuring egg glue protein.The target protein was identified by liquid chromatography-tandem mass spectrometry.Western blot results also showed that the antibodies to His could recognize the recombinant egg glue protein expressed by prokaryotic and eukaryotic proteins with His label.In order to investigate the effect of repeated region on the structure of egg glue protein,circular dichromatography was used to investigate the structural characteristics of egg glue protein.The results showed that high protein concentration promoted the random coils of egg glue protein to ?-helices and finally formed ?-turn structure.The structure of recombinant egg glue protein is very stable at different temperatures and p H,and the content of ?-turn structure can also be increased with the increase of heat.Infrared spectra showed that the ?-turn content of recombinant egg glue protein was higher in eukaryotic expression,and high protein concentration or high temperature was conducive to the transformation of the conformation of egg glue protein from irregular structure to ?-turn.The series of ?-turn can form the ordered conformation of ?-helix,which is considered to be a highly elastic structure,and is also one of the important reasons for the adhesive properties of egg glue protein..3.Analysis of EGP Adhesion Property of SilkwormGlycolysis was performed on repeated region proteins and egg surface eluted proteins expressed in prokaryotic expression and eukaryotic expression systems.It was found that egg surface proteins extracted from egg surface and eukaryotic egg glue protein could be stained with red bands by Schiff base,indicating that both natural and eukaryotic recombinant egg surface proteins have glycolylation modifications.By LC-MS/MS identification,we detected the glycosylation sites and types of natural egg glue protein.It was found that natural egg glue protein contained N-glycosylation modifications and a large number of O-glycosylation modifications,which was very consistent with the properties of sticky proteins.In order to explore the adhesion properties and adhesion mechanism of egg glue protein,we tested the bond strength of eukaryotic and prokaryotic recombinant egg glue protein by universal tensiometer.It was found that the strength of eukaryotic expression of egg glue protein with glycosylation modification was up to 0.8MPa,which was significantly higher than that of prokaryotic expression of recombinant egg glue protein without glycosylation modification(0.2 MPa).Therefore,glycosylation modification could significantly improve the bonding property.In addition,we speculate that laccase,transglutaminase and other cross-linked enzymes can directly catalyze the formation of covalent bonds,which is related to the stickiness of egg glue protein.Since transglutamase catalyzes the formation of isopeptide bonds between glutamine and lysine,which is also consistent with the presence of a large number of Q and K in the amino acid sequence of egg glue protein,we speculated that transglutamase exists in colleterial glands to help the cross-linking of egg glue protein.Based on colleterial gland transcriptome data,we found three genes labeled as transglutaminase and compared them with the sequence of transglutaminase in guinea pigs to confirm the presence of transglutaminase in colleterial glands.SDS-PAGE showed that natural egg glue protein,eukaryotic expressed recombinant egg glue protein and prokaryotic expressed recombinant egg glue protein could catalyse the cross-linking of egg glue protein.The bond strength of the recombinant protein and the natural protein before and after crosslinking was detected,and it was found that the bond strength of the cross-linked eukaryotic recombinant protein could reach 2.2MPa,which was better close to the bond strength of the natural egg glue protein at 2.6MPa,indicating that when silkworm secreted egg glue protein to fixation the ovum vastal surface,Transglutaminase plays an important role in promoting the cross-linking and curing effect of egg glue protein and can improve the viscosity of egg glue protein. |
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