Study On Physiological Indicators Response To Powdery Mildew Of Apple And Screening Of The Resistant Gene Based On Transcriptome Analysis

As an economic fruit,apple（Malus domestica Barkh）,was widely grown in the world.In recent years,apple industry plays a more and more important role in developing countryside in our country.However,powdery mildew caused by the living obligate parasitic Podosphaera leucotricha causes significant economic losses to apple production around worldwide.However,the molecular mechanism of apple’s resistance to powdery mildew is still not completely clear.And apple’s resistance genes to powdery mildew still needs to be further explored and identified.In view of this,this study explores the response mechanism of powdery mildew of apple,using the high quality new cultivar of‘Ruiyang’based on transcriptome analysis.Systematically study of the differentially expressed genes（DEGs）in different period of infection has been made,and related physiological indexes of infected leaves assay and observation of morphology were carried out.The main research results obtained are as follows:1.To gain insight into the molecular basis of PM infection and disease progression,RNA-based transcriptional profiling（RNA-seq）was used to identify differentially expressed genes（DEGs）in apples following inoculation with P.leucotricha.Four RNA-seq libraries were constructed comprising a total of 214Gb of high-quality sequence.1,159DEGs（661 up-regulated and 629 down-regulated）have been identified according to the criteria of a ratio of infection/control fold change>2,and a false discovery rate（FDR）<0.001.The majority of DEGs（815）were detected 12 h after inoculation,suggesting that this is an important time point in the response of PM infection.2、Gene annotation analysis revealed that DEGs were predominately associated with biological processes,phenylpropanoid biosynthesis,hormone signal transduction and plant-pathogen interactions.Genes activated by infection corresponded to transcription factors（e.g.,AP2/ERF,MYB,WRKY and NAC）and synthesis of defense-related metabolites,including pathogenesis-related genes,glucosidase and dehydrin.To identify DEGs associated with hormonal response in the apple leaves infected with PM,the hormone signal transduction pathways were analyzed.To confirm the reliability of expression levels obtained from RNA-seq transcriptome,quantitative RT-PCR（qRT-PCR）was used to analyze expression of 12 DEGs specifically involved in BR signaling and plant-fungal interactions.These results indicate the reliability of the RNA-seq data.3.After inoculation with apple powdery mildew,trypan blue,DAB and NBT staining were carried out on the leaves infected by powdery mildew at 0,3,5,7,10 and 15 days,respectively.It was observed that leaf tissue staining gradually deepened,and the number of dead cells,H₂O₂ and reactive oxygen species produced gradually increased with the extension of infection time.Acquisition by powdery mildew infection 0,1,3,5,7,10 days after apple leaf fresh samples,determination of the apple leaf proline,chlorophyll,soluble sugar and malondialdehyde content,antioxidant enzymes（POD,SOD,CAT）andβ-1,3-glucanase activity determination,comparing the indicators for the response of the pathogen,the results found them with powdery mildew infection are regularity of change.The results showed that the accumulation of hydrogen peroxide,reactive oxygen species,dead cells,proline and malondialdehyde in infected plants,as well as the increase of related enzyme activities,reflected the significantly enhanced resistance of apple leaves to living parasitic bacteria.Through the expression analysis of related genes,the results showed that the expression of genes was consistent with the changes of relative physiological indexes.4、Based on the results and those of previous studies,a possible molecular network underlying the defense response in apple leaves to PM infection was diagrammed.The process can be divided into four stages:signal sensing and transduction,transcriptional regulation,the synthesis of downstream functional proteins and the production of metabolite.Some physiological indexes of the plant also changed correspondingly,which enhanced the resistance of apple to powdery mildew.