Identification Of Mulberry Protein With Antidigestive Stability And Expression Patterns Of Genes Encoding Mulberry AS Protein

Plant proteins with Antidigestive Stability(AS)are a type of defense protein that remain stable and intact in the gut lumen of phytophagous insects and eventually in frass.In general,the production of AS proteins in plants is induced by insect feeding.Mulberry is not only a perennial tree with economic value,but also an essential feedstuff for silkworms.Consequently,mulberry trees play crucial roles in forest and sericulture industries.Therefore,it is important to study insect resistance mechanisms in mulberry.To date,mulberry AS proteins,which are closely related to defense against insect herbivores,have not been systematically identified and analyzed.Here,we identified AS proteins in silkworm frass using liquid chromatography-tandem mass spectrometry technology,and monitored induction of their expression by silkworm feeding.This information provides supporting data to understand how mulberry trees defend against insect herbivores.The main results can be summarized as follows:1.Identification and functional classification of mulberry AS proteinsMulberry AS proteins were extracted from silkworm frass and analyzed in using a shotgun strategy combining one-dimensional SDS-PAGE and liquid chromatography-tandem mass spectrometry.A total of 152 mulberry AS proteins were matched by searching the mulberry protein database,with some proteins corresponding to more than two unique peptides.The classifications of proteins included energy,metabolism,protein destination and storage,secondary metabolism,signal transduction,transcription,transporters,cell growth and division,cell structure,intracellular traffic,disease and defense and other proteins.More than 26.3% of identified proteins were disease-and defense-related proteins.Comparative analyses among mulberry AS proteins,the leaf and the latex proteome showed that 122 proteins could be detected in both mulberry frass and mulberry leaves.Only 24 mulberry proteins were found only in silkworm frass.2.Expression analysis of genes encoding mulberry AS proteinsThe transcript levels of genes encoding mulberry AS proteins in Morus notabilis latex and leaf were determined using q RT-PCR analyses.Fifty genes showed higher expression in the latex and 95 genes showed leaf-biased expression.In functional annotation analyses,the top three classifications of gene expressed in mulberry latex were disease and defense,energy,and protein destination and storage.For the genes expressed in leaves,the top three classifications were energy,metabolism,and disease and defense.Next,we identified AS protein genes whose transcription was induced by silkworm feeding treatment in M.notabilis leaves.The data indicated that 80 mulberry AS protein genes were significantly up-regulated within 24 h of silkworm feeding,and 51 mulberry AS protein genes were significantly down-regulated.These data provides important insights into the functions of mulberry AS proteins.