Regulation On Sphingolipid Metabolism And Ceramidase By Nitric Oxide-Cold Stress Signalling Cross-talk

Sphingolipids are primary components of membrane lipids,which are necessary for the normal fluidity of cell membrane.As a kind of signal molecular,sphingolipids participate in a number of signal transduction processes,such as cellular differentiation,growth,senescence and programmed death.Ceramide is the central product of sphingolipid metabolism,and will be hydrolyzed to sphingosine and fatty acid by ceramidase.It is able to transmit signal of apoptosis and growth retardation.Nitric oxide?NO?is an important signal molecular that it plays important roles in regulating numbers of physiological processes,delaying senescence,alleviating chilling injury.Researches have showed there are NO-cold stress signalling cross-talk?NSC?in plant.Nowadays,studys about the regulation on fruit chilling injury by NO mainly focus on the effects of NO on alleviating chilling injury and imporving the fruit quality during the storage.However,there are few researches about the mechanism of membrane lipids recepting cold signal and the effects of NSC on composition and metabolism of membrane lipids.In this study,total proteins of ‘Feicheng' peach were extracted and detected by iTRAQ technique.A total of 4776 proteins were identified which were involved in 135 metabolic processes.The difference on storage temperature was more likely to cause difference of protein expression.GO analysis and COG analysis showed that protein metabolism and sugar accumulation were the main physiological activities of peach fruit during postharvest storage.There were 31 proteins detected in sphingolipid metabolism,16 of which were differential proteins.These differential proteins included ?-galactosidase,serine palmitoyltransferase,neutral ceramidase,phosphatidic acid phospholipase and so on.Based on the results of iTRAQ,PPCD_ase?Prunus persica ceramidase?was selected as the target of the follow-up study.The full length of PPCD_ase gene was cloned by homology cloning technique and rapid amplification of cDNA ends?RACE?.It was 2784 bp,total of 2337 bp open reading frame encoding 778 amino acids.The results of blast in NCBI and simulation of three-dimensional structure indicated that the gene was similar to neutral ceramidase genes of plum and pear.The physical and chemical properties analysis showed that PPCD_ase was a hydrophilic protein with strong stability.The dimensional structure simulation showed PPCD_ase contained 16 ?-helix and 25 ?-fold.S-nitrosation prediction showed there were 6 S-nitrosation sites within PPCD_ase.The in vitro expression of PPCD_ase recombinant protein was carried out with the prime designed according to the full length of PPCD_ase gene,and the protein was purified by nickel column purification method.Electrophoresis result showed that the protein appear in the 112 KDa band,which indicated protein was gained successfully.The results of qPCR showed exogenous NO treatment could delay the decrease of PPCD_ase gene expression,especially during cold storage.