The Proteomics Analysis Of Downy Mildew Infection Related In Cucumber Leaf Intercellular Space

In this study,the resistant germplasm"NW088" and susceptible germplasm "Changchun mici" were used as test material and were inoculated by spraying.The intercellular spaces proteins were separated and identified by differential proteomics technology.We tried to found protein associated with resistance to downy mildew bio-informatics analysis and provided the important information for cucumber breeding in disease resistance.Control and inoculated leaves after 1,2,4 d were taken to extract the intercellular space fluid by vacuum infiltration.The centrifugation speed was 1000g and centrifugal time was 10 min.To determine the intercellular space fluid purity,the malate dehydrogenase activity in the intercellular space fluid was measured.The intercellular space protein samples were prepared by precipitation with trichloroacetic acid/acetone.In this way,we got a good repeatability with clear background 2-DE pattern more conducive to the intercellular space differential proteomics analysis.We analysed the intercellular spaces protein 2-DE map of resistant germplasm"NW088"before inoculation and vaccination 1,2,4 days after.24 protein spots were found,of which there were 15 spots after vaccination compared with the control up-regulation,more than 1.5 times the control;9 protein spots were down-regulated,was 0.67 times.We also analysed the intercellular spaces protein 2-DE map of susceptible germplasm "Changchunmici "before ino-culation and vaccination 1,2,4 days after.25 protein spots were found(some with NW088 repeat),of which there were 15 spots up-regulated after vaccination compared with the control,more than 1.5 times the control;10 protein spots were down-regulated,was 0.67 times.We analysed the intercellular spaces protein 2-DE map of the "NW088" and"Changchunmici" at different time points,35 differentially expressed protein spots were found(some spots and repeat earlier)of which 22 proteins were up-regulated and 13 down-regulated.After the above analysis,we found 49 protein spots by MALDI-TOF-MS-MS analysis.46 protein spots were identified and the identification success rate was 94%.SignalP,TMHMM,TargetP,SecretomeP-1.0 programs were use to predict and locate the identified proteins,26 protein spots of 46 identified protein spots belonged to the intercellular space proteins,including 9 proteins containing a signal peptide secreted by the traditional secretory pathway and 17 leaderless peptide protein without signal peptide,65%of the intercellular space proteins is secreted by non-traditional secretory pathway.We analyzed the function of the identified protein in intercellular spaces,found that 35%of the proteins involved in the metabolism of carbohydrates,8%of the protein involved in the redox reaction,15%of the proteins involved in protein metabolism,8%of the protein involved in cell wall biosynthesis and degradation,15%of the proteins involved in energy metabolism,8%of the proteins involved in lipid metabolism,8%of the protein involved in other metabolic processes,in addition to 3%of the protein of unknown function.We remove three proteins that were chitinase enzyme,cationic peroxidase and esterase precursor from the 26 identified proteins in the intercellular spaces randomly to analyze their differential expression at the transcriptional level by qPCR.We found they had the same trend in protein expression level and the transcription level.