Effect Of Intracellular Proteins Expression Of Colletotrichum Gloeosporioides In Response To Borate Based On Proteomics Approach

Anthracnose is a kind of fungal disease which have a worldwide distribution.It is one of the most serious diseases in mango fruit caused by Colletotrichum gloeosporioides(Penz.).As the pathogen can latently infect fruitlet of mango,postharvest diseases always induced the mature fruit.Recently,application of chemical fungicide is the major approach to control the anthracnose at the peroids of preharvest and postharvest.However,the negative effect of chemical fungicide is more and more obvious,for example,damage to the environment and drug resistance and so on.So,the researchers want to explore a new way to control the fruit diseases.Boron is a kind of important trace element for plant and is widespread in the soil.Borate is a safe and effective soil fertilizer,it can improve crop quality and yield.Hence,borate,a new type of anti-fungi,is used to control postharvest diseases of fruits and has great potential.Despite it is reported in the literature about the borate to control postharvest anthracnose of mango,few information regard to the mechanism that borate inhibit pathogenic fungi to use a proteomic strategy.This study is based on a comparative proteomics,using two-dimensional electrophoresis(2-DE)and MALDI-TOF-TOF-MS echnology to study the impact of intercellular proteins of Colletotrichum gloeosporioides under normal condition and borate stress.And quantitative real-time PCR assay was used to investigated the correlation between transcription and translation.Firstly,obtain different protein maps by 2-DE which different concentration of potassium tetraborate(OmM,5mM and 7.5mM)were treated on anthracnose conidium at 36h and 48h.Then by means of MALDI-TOF-TOF-MS echnology,analysis and identified of differentially expressed proteins.Thus,the metabolic system network will be infered about the impact of intercellular proteins of Colletotrichum gloeosporioides under normal condition and borate stress.Twelve proteins were selected from metabolic system network to analyse variation of expression quantity of gene.The results are as follows:1.Treated with different concentrations of potassium tetraborate(OmM,2.5mM,5mM,7.5mM and lOmM)in anthracnose and measured the colony size.Select the concentration of 5mM and 7.5mM of potassium tetraborate to dispose the conidium of Colletotrichum gloeosporioides used for protein extraction and two-dimensional electrophoresis experients.2.1t was detected about 900 protein spots by 2-DE and PDQuest software(Ver8.0,Bio-Rad).At the same time,145 differentially expressed protein spots were analyzed.By MALDI-TOF-TOF-MS 77 protein spots were successfully identified.3.Through the expression analysis of 77 differentially expressed protein spots,31 protein spots were significantly up-regulated.28 protein spots were significantly down-regulated and 18 protein spots were both significantly up-regulated,also significantly down-regulated.4.77 differentially expressed proteins can be divided into 12 functional types.they include energy metabolism(12.6%),redox homeostasis(19.5%),signal transduction(5.8%),cell rescue(1.2%),proteolytic proteins(9.2%),protein biosynthesis(18.4%),protein folding and assembly(6.9%),carbohydrate metabolism(3.4%),amino acid metabolism(6.9%),nucleotide metabolism(2.3%),cellular processes(8.0%)and unclassified(5.8%).At the period of 36h,functional classification include energy metabolism(13.5%),redox homeostasis(18.9%),signal transduction(6.8%),cell rescue(1.4%),proteolytic proteins(10.8%),protein biosynthesis(21.6%),protein folding and assembly(6.8%),carbohydrate metabolism(1.4%),amino acid metabolism(8.1%),cellular processes(6.8%)and unclassified(4.0%).At 48h,functional types were energy metabolism(14.1%),redox homeostasis(17.2%),signal transduction(3.1%),cell rescue(1.6%),proteolytic proteins(7.8%),protein biosynthesis(18.8%),protein folding and assembly(7.8%),carbohydrate metabolism(4.7%),amino acid metabolism(4.7%),nucleotide metabolism(3.1%),cellular processes(10.9%)and unclassified(6.2%).Among them,large proportion at energy metabolism,redox homeostasis and protein biosynthesis.5.Under borate exposure,energy expenditure are enhanced in conidium of Colletotrichum gloeosporioides.Reduced peroxide scavenging capacity and enhanced oxide generating.Signal transduction and proteolytic proteins are decreased under borate exposure.Whereas accelerated biosynthesis of proteins and protein folding and assembly.Some pathways also showed a trend of increased in borate stress like amino acid metabolism and nucleotide metabolism.In addition,potassium tetraborate are also affected carbohydrate metabolism,cell processes and cell rescue.6.Twelve proteins were selected from metabolic system network to identify by quantitative real-time PCR.It is find that protein and gene expression had a good relativity but individual inconformity.So,it is necessary to detect on transcription after detection of proteins.