Regulation Of Resveratrol O-methyltransferase Gene In Pterostilbene Defensing The Sour Rot Of Wine Grape

Recently,sour rot is one of the main diseases which were found in the process of fruit ripening,storage and transportation,which not only causes the occurrence of rotten fruit in the postharvest period,but also seriously affects the quality of grape and wine.At present,chemical fungicides are main methods to prevent sour rot.While it would not only produce resistant strains,but also would endanger human health.In recent years,phytoalexin has attracted more attention from people.Pterostilbene is an important phytoalexin in resisting external biological or abiotic stress.Previous studies found that pterostilbene show the evident antifungal activity against Geotrichum citri-aurantii,and it could inhibit the activity of G.citri-aurantii at the pharmacological concentration in vitro.However,it was not well known that the mechanism of pterostilbene defence to G.citri-aurantii-stress in grape.On the base of the previous studies,our investigation focused on the defense mechanism of pterostilbene against G.citri-aurantii.The secondary metabolites of grape defensing sour rot were separated,and then the identification of the secondary metabolites was performed.After that the key gene of defense was cloned and verified in vivo and in vitro.The results included the following four aspects:1.The effect of pterostilbene inhibit G.citri-aurantii was studied in vitro.The results showed that with the increase of pterostilbene concentration,the inhibitory effect was significantly enhanced,when the concentration of pterostilbene is 400 g/mL,the inhibition rate reached 79.91%.In order to explore pterostilbene defense sour rot in grape,taking grapes as the main plant materials,the direct contact method was used to make grape infected by G.citri-aurantii.Then the extent of grape decay was evaluated by the physiological level.Results showed that grape began to decay,accompanied by the smell of sour rot at 48 h after infected.Grapes rotted severely at 72h after infected.To resist the infection of diseases,secondary metabolites were synthesized in grape.Two secondary metabolites,resveratrol and pterostilbene,were separated and identified by ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry(UHPLC-QQQ-MS2).Furthermore,UHPLC-QQQ-MS2 was used to monitor the content change of two secondary metabolites in the process of defense,indicating that infection of G.citri-aurantii would induce the content of resveratrol and pterostilbene increasing.2.Using molecular biological sequencing technology and the existing grape expressed sequence tags(EST)database and grape genome database,we obtained the key gene of pterostilbene defense,the resveratrol oxy methyltransferase gene(WgREOM),and the sequence analysis is carried out.The open reading frame(ORF)of WgREOM cDNA is 1116 bp which encodes 305 amino acids with a molecular mass of about 34.4 kDa and an isoelectric point of 6.52.Analysis of holistic relationships indicated that WgREOM and european grapes resveratrol oxy methyltransferase gene(VvROMT)belong to the same subfamily,but closed to the phylogenetic location of lichenol oxy-methyltransferase(VvCOMT).This putative WgREOM cDNA from wine grapes shared 95.86%nucleotide sequence similarity with european grape VvROMT.The sequence analysis revealed that Chinese rose OOMT1,Castor oxy-methyltransferase(RsOMT)and oxy-methyltransferase from Iris holland(IhOMT)had 65.61%,62.10%and 30.57%nucleotide sequence similarity with the candidate REOM,respectively.3.Function of objective gene(WgREOM)was verified by fluorescent quantitative polymerase chain reaction(qPCR).Using G.citri-aurantii to infect grape,the response of objective gene(WgREOM)defensing pathogenic fungi was detected by qPCR,which showed that the expression of WgREOM gene change with infected time,presenting the trend of increasing first and then decreasing.The expression of WgREOM gene reached the maximum at 24h after infected,then decreased at 48h after infected.WgREOM gene almost doesn't expressed at 72h after infected.4.The REOM protein was verified in vitro by eukaryotic expression system and high performance liquid chromatography(HPLC).First of all,the eukaryotic expression vector pPIC9K-REOM was constructed.After identified by PCR and enzyme restriction,the vector containing the target gene WgREOM was transformed into Pichia pastoris expression system.Then,its expression was induced,and corresponding protein was detected by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis(SDS-PAGE)and Western blot,a molecular mass of the protein was about 34.4 kDa.Afterwards,adding reaction substrate resveratrol to buffered glycerol-complex medium(BMGY)which contained recombinant yeast.Pterostilbene has been detected by HPLC,which demonstrated that WgREOM has the oxygen methylation function which could catalyze the synthetize of pterostilbene in vitro.