Screening And Protein Interaction Network Analyzing Of Heat Resistant Key Protein In Alicyclobacillus Acidoterrestris

Alicyclobacillus acidoterrestris,a thermophilic and acidophilic bacterium,can survive undertraditional pasteurization with unique viability,which caused serious economic losses.How to control contamination of A.acidoterrestris had become a common concern in the food research field and fruit juice industry.It is crucial of pollution solved and hazard control to investigate the heat-resistant mechanism.Label-free quantification,bioinformatics analysis and qRT-PCR was used to screen differentially expressed proteins of A.acidoterrestris under heat stress,to study the molecular functions of differential proteins and the metabolic pathways they involved and to screen the key regulatory proteins response to heat stress from the large numbers and enriched differential expressed proteins;the heat resistant key protein network was predicted and validated by STITCH database and yeast two-hybrid technique,which revealed the molecular function and interaction mode with interaction proteins of heat-resistant key proteins in A.acidoterrestris under heat stress,elucidated the molecular mechanism of key proteins regulating the A.acidoterrestris response to heat stress.The main findings are as follows:1.By colony counting method and environmental scanning electron microscopy,it was found that there was no significant change in the number of colonies when the treatment was carried out for 5min at 65?,and the surface of the cells was slightly damaged,it is inferred that the cells at this treat condition are active and the expression of protein is change different.Therefore,65?,5 min were selected as the heat-treat condition.2.Totally,545 differential expression proteins were respectively identified at heat stress of 65? for 5 min,of which 258 proteins were up-regulated and 287 proteins were down-regulated.These significantly changed proteins were mapped to 100 pathways and some of them were mostly related to protection or repair of macromolecules such as proteins and DNA,cell wall formation,which indicated that these proteins might play crucial roles in response to heat stress.The KEGG pathway analysis combined with protein functional analysis and further validation at mRNA level suggested that A.acidoterrestris sensed the temperature rise in environment through alterations in the secondary structure of DNA and RNA molecules.The biosynthesis of antibiotics pathway and the ribosomes might be involved in signal transduction in heat stress and further trigger a large number of proteins playing a critical role in the regulation of heat stress in A.acidoterrestris.3.Heat-shock proteins were chosen in this study to obtain the targets of the protein-protein information 37 totally through the STITCH databases retrieval.Protein interaction network were established,module analysis and function annotation of network were analyzed.It was found that the heat-shock protein biological processes involved are related to stress response,accompanied with biosynthesis of macromolecules and nucleic acid metabolism,which further verified the conclusion of qRT-PCR,and the bait protein DnaK for yeast-hybrid was screened.4.The heat-resistant key protein was used as material to generate the first strand cDNA bySMART cDNA synthesis technology with the template mRNA,long distance PCR was used to amplify double-strand cDNA(ds cDNA)which normalized by duplex-specific nuclease(DSN).The normalized cDNA was removed the short cDNA and purified.The purified cDNA and vector pGADT7 were co-transformed into E.coil to generate a normalized yeast teo-hybrid cDNA library.Harvested library was more than 1×10~6 CFU,average length of the insertion was more than 900bp and the positive rate was more than 95%.The library had high quality and can be used in yeast two-hybrid.The quantity of interaction with DnaK is abundant,and most of them are heat shock protein.Dnak is involved in the heat stress response,which is consistent with the proteomics.Therefore,the DnaK is selected as the bait protein for yeast two-hybrid.This research reveals the physiological adaption mechanism of A.aciodterrestris on heat resistance,which would establish an experimental and theoretical foundation for food preservation,the research of microbial stress resistance and development of special industrial bacteria resistant to high temperature.