Termites Gut Proteomics Research In Different Feed

Termites,as part of an ecosystem circulation,play an irreplaceable role on the decomposition of dead trees,weeds and fallen leaves compared with bacteria,fungi,its ability to digest the lignocellulose is also remarkable.Screening high specificity of cellulase from termites and reasarching lignocellulose degradation mechanism of termite gut are very significance to the efficient utilization of lignocellulose.The research object is Reticulitermes perilucifugus,feed them in three different diets,changes of protein components in different parts of gut are compared and analyzed by two-dimensional electrophoresis technology with MALDI-TOF/TOF and Q Exactive,determine the workers gut protein composition,to further understand the termites nutrition digestion and absorption and its regulation mechanism.Results are followed:1)Under the feeding of pinewood,termites hindgut Q Exactive sequencing showed that there are 2155 proteins involved in the metabolic process in termites gut,107 proteins directly involved in carbohydrates decomposition process;main metabolic pathways contain carbon metabolism,ammonia acyl biosynthesis,the biosynthesis of amino acid,fatty acid degradation,purine metabolism,glycolysis,and sugar,oxidative phosphorylation,new pyruvate metabolism,methane metabolism,RNA transportation,endocytosis,endoplasmic reticulum protein processing,etc.These metabolic pathways conform to the termites gut protein characteristics,especially the carbon metabolism,glycolysis and sugar freshmen will be our focus.Termites in lignocellulose degradation enzyme contains glucanase,cellobiose hydrolysis enzyme,beta glucoside enzyme,alpha glucoside enzyme,hemicellulase,peroxidase.No lignin degradation enzymes found in termites gut in the results of Q Exactive sequencing,we speculate that lower termites can,t utilize lignin well,most of lignin was excreted as feces.2)This experiment used Total Protein Extraction Kit for Microbes with Thick Cell Walls with TCA acetone to extract total protein of termite gut contents,optimized the working concentration and time of SDS,DTT and urea reagent in the process of protein extraction,determined Protein usage and electrophoresis conditions of two-dimensional electrophoresis isoelectric focusing step.Finally we established the method of termites gut contents total protein two-dimensional electrophoresis.3)two-dimensional electrophoresis results showed that: promesenteron discernible protein gel points are obviously more than hindgut,among three different diets;the number of discernible protein gel points of promesenteron and hindgut from more to less is pinewood> filter paper> cornstalk;there are 21 same protein gel points existed in all six gel draw,their abundance are very high although the expression of each protein are different;the electrophoregrams of promesenteron protein in filter paper and cornstalk diets are similar,and the electrophoregrams of hindgut protein are similar among three diets.Change of feeding conditions cause great influences on protein types,expression of termites gut protein.Differences between promesenteron and hindgut protein suggested that the different positions of the guts has its unique function.At the same time,different feed cause great influences in promesenteron,but the influences in hindgut is not significant.4)Different protein gel points among three diets was sequenced,results showed that these proteins are mainly catalytic activity protein,immune regulatory proteins,cells and signaling proteins.Among these proteins,Heat shock 70 k Da protein 、Protein lethal(2)essential for life 、 Chaperone protein Dna K play a variety of physiological functions,make the cell metabolism running smoothly,expression level of them are increased under the pinewood diet.That reveal termites were in a state of environmental stresses under the condition of corn stover and filter paper feeding.The proteins with catalytic activity,including amino acid metabolic enzymes,pyruvate metabolism enzyme,carbon metabolic enzymes,TCA cycle enzyme,glycolysis and sugar related metabolic enzymes,new nitrogen metabolic enzymes,cellulase and some other catalytic activity of the protein,expression level of these proteins are also increased under pinewood diet to provide enzymes for energy metabolic and maintaining the gut environment.These enzymes are mainly termites own endogenous protein,only Superoxide dismutase [Mn],Chaperone protein Dna K,Threonine-t RNA protein ligase are generated by gut symbiotic microorganisms.Most different protein gel points under corn stalk and filter paper diets may be new protein because of can’t match the database.Termites under three different diets,found in pine breeding and straw for different glucanase cutting inside,and found only in front of the straw feed and filter paper feeding in the intestine of Mn superoxide dismutase(sod),beta glucoside enzyme.To show that the termites chew food,after the former gut already have full capacity for degradation of lignocellulose,and then in the hindgut termite itself and microbes secrete more abundant hemicellulase,cellobiose hydrolysis enzyme,beta glucoside enzyme,fully degradation of cellulose and hemicellulose,matter and energy.