Mining And Research Of Protein Molecular Markers Related To Quality Evaluation Of Silkworm

Bombyx Batryticatus is a dried body killed by the larvae of Bombyx mori Linnaeus,a silkworm moth family,infected(or artificially inoculated)with Beauveria bassiana(Bals.)Vuillant.According to the research of literature,it is required that Bombyx Batryticatus should have the following qualities:"big and no maggots","straight/thick strips","hard quality"and"black and bright belly breaking".In recent years,the demand for it has increased year by year.There are fake products on the market,such as lime water mixed with stiff silkworms,weight-added stiff silkworms,green stiff silkworms,yellow stiff silkworms,drum fried stiff silkworms,which cause market confusion and affect the safety and effectiveness of clinical medication.In 2015 edition of Chinese Pharmacopoeia(Part I),only five items of impurities,water,total ash,acid insoluble ash and aflatoxin were examined to identify and evaluate the quality of Bombyx Batryticatus.Moreover,the effective or effective ingredients in Bombyx Batryticatus are not clear,and its modern pharmacological research is not deep enough,which makes the quality standard of its imperfect and the efficacy of its effective.It can't be guaranteed,which limits the utilization of Bombyx Batryticatus resources.Protein is one of the main components of Bombyx Batryticatus,and it has interspecific specificity and stability.The aim of this study is to explore the optimal extraction process of alkaline soluble protein from Bombyx Batryticatus.The protein composition of Bombyx Batryticatus was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and acetone grading precipitation(AFP)/Tricine-SDS-PAGE technology.Its protein composition,analysis of differentially expressed protein molecules of different quality silkworms,provides a theoretical basis for tapping protein molecular markers related to the evaluation of silkworm quality and further elucidating the molecular mechanism of its quality formation.The research content of this paper mainly includes:1.The extraction process of alkali soluble protein from Bombyx Batryticatus was established.Using 0.1 M Tris-HCl buffer as solvent to extract protein from Bombyx Batryticatus,the effects of p H,ratio of liquid to material,extraction temperature and extraction time on protein extraction rate of Bombyx Batryticatus were investigated to find the best single factor level.On this basis,according to the central experimental design principle of box behnexn,29 experiments were designed with 4 single factors as independent variables and the extraction rate of protein as response value.The results showed that the optimal extraction conditions were p H 8.0,the ratio of liquid to material58:1(m L/g),temperature 40?,extraction time 205 min.the protein extraction rate was 2.29%.2.To establish and optimize the SDS-PAGE fingerprint of Bombyx Batryticatus protein.0.1 M HAc-Na Ac buffer(p H 4.8),0.1 M Tris-HCl buffer(p H 8.0),H₂O and40%Et OH were used to extract protein from it.The protein was analyzed by SDS-PAGE.The effects of protein loading,separation gel concentration and gel dyeing method on protein fingerprints were analyzed.The results showed that the amount of acid soluble,alkali soluble,water-soluble and alcohol soluble was 20?g.Silver staining method is more sensitive than coomassie brilliant blue staining method,and more different protein bands can be observed.3.The differentially expressed protein molecules in the selected and the unselected Bombyx Batryticatus were analyzed by LC-MS/MS and bioinformatics.The improved AFP/Tricine-SDS-PAGE method was used to display the differentially expressed protein molecules,which were identified by LC-MS/MS.A total of 190 differentially expressed proteins were found,distributed in cells,cells and protein-containing complexes,with catalytic,antioxidant,in vitro binding and transport functions,and these differentially expressed molecules mainly involved in microbial metabolism and carbon metabolism signal pathway,etc.revealed that the above molecules and signal pathway may have an important impact on the formation of the quality of Bombyx Batryticatus,and also provided an important reference and basis for the establishment of its quality evaluation technology based on specific protein molecular markers.