Mining And Research Of Protein Molecular Markers Related To The Origin Of The Silkworm

Bombyx batryticatus is a dried body that was dying from the larvae of the Bombycidae insect silkworm(Bombyx mori L.)infected with Beauveria bassiana(Bals.)Vuillant.Bombyx batryticatus was originally published in"Shennong's Herbal Classic",which has a long history of application,and has the effects of dispelling wind and convulsing,resolving phlegm and disintegrating knots.Bombyx batryticatus is a kind of medicinal material with high production area,and ecological factors of different production areas will affect the quality of Bombyx batryticatus.Therefore,it is necessary to establish the traceability technology of the origin of Bombyx batryticatus to ensure the quality of the Bombyx batryticatus medicinal materials and regulate the market of medicinal materials.This article aims to explore the optimized extraction process of Bombyx batryticatus protein and study the composition of Bombyx batryticatus protein with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and improved acetone fractionation precipitation(AFP)/Tricine-SDS-PAGE technology.The main results are as follows:1.Establishing an optimized extraction process of Bombyx batryticatus prolamins,then construct its fingerprint by using SDS-PAGE,HPLC,and FTIR.The results showed that the optimal extraction process conditions for Bombyx batryticatus prolamins were 1:24.22 g·mL^-1 material-liquid ratio,ethanol concentration 38.73%,temperature 35?,extraction time 3.94 h,and the extraction rate was 0.5402%.The results of SDS-PAGE showed that the molecular weight of Bombyx batryticatus prolamins was concentrated in 16.7?36.2 kDa;The results of HPLC showed that the retention time of main Bombyx batryticatus prolamins was 2.048 min and 3.354 min,and their contents accounted for 31.23%and 32.93%of the total protein,respectively;The results of FTIR showed that Bombyx batryticatus prolamins had major absorption peaks at 2927.71,1631.71,1457.24,and 1310.71 cm^-1.2.Establish an optimized protein SDS-PAGE fingerprint for the identification of Bombyx batryticatus medicinal materials.In this study,0.1 M HAc-NaAc buffer(pH4.3),0.1 M Tris-HCl buffer(pH 8.6),ddH₂O and 40%ethanol were used to extract the Bombyx batryticatus proteins from Yunnan and Sichuan,and the SDS-PAGE analysis showed that:When the protein loading was 20?g and the gel concentration was 14%,the protein fingerprint of Bombyx batryticatus reached the best resolution.The sensitivity of the silver nitrate staining method is significantly higher than that of the coomassie brilliant blue method,and Tricine-SDS-PAGE is more suitable for analyzing small molecular proteins in Bombyx batryticatus.3.Excavate the differentially expressed protein molecules in Bombyx batryticatus from Yunnan and Sichuan,and analyze them by LC-MS/MS and bioinformatics.In this study,0.1 M HAc-NaAc buffer(pH 4.3),0.1 M Tris-HCl buffer(pH 8.6),ddH₂O and40%ethanol were used as extraction solvents.The improved AFP/Tricine-SDS-PAGE was used to analysis differentially expressed protein molecules from Yunnan,Sichuan.The results showed that a total of 16 different protein bands were found.Using LC-MS/MS to identify the differentially expressed molecules of Bombyx batryticatus protein,and then we obtained 273 differentially expressed molecules with scores?1.3.At the same time,this study also conducted bioinformatics analysis on differentially expressed molecules.The results show that these differentially expressed molecules are mainly distributed in cellular organisms,nucleus and cytoplasm,and their molecular function classification mainly includes catalytic activity and binding function,and their biological processes mainly focus on cellular processes,metabolic processes,stress reactions,biological Adjustment,etc.The results also showed that the content of carbohydrate metabolism-related proteins accounted for the highest proportion,reaching 25.78%,followed by protein conversion,modification-related proteins and molecular chaperone proteins,reaching 23.83%.And these differential molecules mainly involve the biosynthesis signal pathway of secondary metabolites,microbial metabolism signal pathway,antibiotic biosynthesis signal pathway,carbon metabolism signal pathway,amino acid biosynthesis signal pathway,etc.The above pathways may be important targets for environmental factors in the Bombyx batryticatus producing areas,and have an important impact on the quality of Bombyx batryticatus production in different producing areas.They also provide important references and basis for establishing traceability techniques for Bombyx batryticatus producing areas based on specific protein molecular markers.