| Pears(Pyrus)are deciduous fruit trees native to China.They are mainly divided into four types:white pear,sand pear,ussurian pearand western pear.Among them,’Cuiguan’(Pyrus pyrifolia)is a typical semi-russet pear variety of excellent quality and strong adaptability and widely planted in the drainage areas of the Yangtze River in China,but the process of fruit development coincides with a rainy season and the fruits usually exhibit the semi-russet phenotype,which significantly affects fruit appearance quality,reduce consumers’ acceptance and the economic value.In this study,preharvest bagging treated and untreated ’Cuiguan’fruits were used as experimental materials to characterize fruit semi-russet formation by histo-morphology analysis and metabolite profiling.Through comparative transcriptome analysis,dozens of differentially expressed genes involved in fruit semi-russet formation were identified.Meanwhile,Candidate PRX genes were isolated by RNA-Seq.Enzyme activity assay,subcellular localization and prokaryotic expression were adapted to identify PRX genes regulating pear fruit russet formation.enzyme The results are as follows:1.The morphological changes and quantitative analysis of lignin,cutin and suberin between skins of untreated and bagged treated ’Cuiguan’ pear fruits were clearly identified.During the fruit development of ’Cuiguan’ pear fruits,the occurence of fruit russet mainly focus on the middle and later stages of fruit development.Through microscopy observation and chemical dyeing,it was found that the russeted pear skins are composed of a defective cuticle layer,russet deposited layers and periderm layer and are significantly different from bagging treated pear skins.Quantitative analysis of lignin,cutin and suberin revealed that fruit russeting is the results of decreasing in cutin and increasing in lignin and suberin..The decrease of cutin is mainly caused by the reduction of ω-hydroxy fatty acids(C16-C18),among which the decrease of C16 ω-hydroxy fatty acids is the most obvious;the increase of suberin content is caused by including aromatic monomers(mainly ferulic acid)and the increase of lipid monomers,such as fatty acid(C19-C23),fatty alcohol(C22-C26),α,ω-dicarboxy fatty acid(C16-C22)and ω-hydroxy fatty acid(C22,C26).2.A comparative RNA sequencing analysis wasapplied to detect the differentially expressed genes involved in russet formation of ’Cuiguan’ pear fruit.A total of 1000 differentially expressed genes were screened,including 67 differentially expressed genes related to russet fruit periderm layer formation,including 15 genes related to suberin metabolism,9 genes related to cutin metabolism,8 genes related to phenylpropaniod synthesis,7 genes related to lignin biosynthesis,12 genes related to oxidase and 16 genes related to cell wall modification.Except that the genes related to cutin metabolism are negatively correlated withrusset formation of ’Cuiguan’ pearfruit,the other genes are positively regulating the formation of fruit rust.At the same time,a total of 120 differentially expressed transcription factor distributed in 17 families were also obtained,and the number of of differentially expressed MYB members gene family was significantly higher than other transcription factor members,which might be the key transcription factors involved in russet formation of pear fruit.3.14 differentially expressed PRX genes were isolated through transcriptome sequencing and phylogenetic analysis.These genes showed different expression patterns andPRX8,PRX9,PRX49 and PRX81 showed a positive correlation with pear fruit russet formation and selected as candidate PRX genes for further analysis.The results of subcellular localization showed that the four candidate PRX genes were all located on the cell wall.At the same time,four PRX proteins were purified by prokaryotic expression system,which laid the foundation for subsequent analysis of the affinity of candidate genes to different substrates and identification of their biological functions. |
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