Differential Expression Analysis Of Anthocyanin Biosynthesis Related Genes In Leaves Of Malus ‘Profusion’ Under Rust Stress

When being invaded by insects and pathogens,plant organs carry out direct defense reaction by regulating physiological metabolism,and these physiological metabolites tend to accumulate in the infected area,forming disease spots.When the leaves of Malus ‘Profusion’were infected by crabapple rust(Gymnosporangium yamadai Miyabe),a large amount of anthocyanin would accumulate around the disease spot,forming obvious red patches.At present,there are few reports on the molecular mechanism of anthocyanin formation on the red spots in leaves of crabapple under rust stress.In this study,through the early observation of characteristics in rust-infected leaves of crabapple and germination culture experiment of teliospores,the formation time of red spot was found.After,the green uninfected leaves tissue(UIT)and red rust-infected leaves tissue(RIT)of Malus ‘Profusion’ was used as experimental materials respectively.The anthocyanin related metabolites were quantitatively analyzed by high performance liquid chromatography.Expression levels of structural genes and transcription factors related to anthocyanin biosynthesis were analyzed by transcriptome sequencing combined with real-time quantitative PCR.The biosynthesis mechanism of anthocyanin in RIT of Malus ‘Profusion’ was revealed through the analysis of the changes in the expression levels of anthocyanin related genes and related metabolites in UIT and RIT.The research results are as follows:(1)Anthocyanin and its metabolites were abundant in the RIT of Malus ‘Profusion’.The anthocyanin analysis showed that the content of total anthocyanin(The main type is cyanidin-3-galactoside chloride)in RIT is 18.50 times as much as that in UIT.The contents of naringenin,dihydroquercetin,catechin and epicatechin,which are the main by-products in the anthocyanin synthesis pathway in RIT,were respectively 1.57,2.38,2.59 and 4.89 times as much as that in UIT.(2)A total of 47372 high quality gene sequences were obtained by RIT and UIT transcriptome sequencing.Furthermore,the total 10045 differentially expressed genes(DEGs)have been found,including 6021 up-regulated genes and 4024 down-regulated genes(RIT vs.UIT).Twelve structural genes related to anthocyanin metabolism were screened from the DEGs,including phenylalanine ammonia-lyase(PAL),chalcone isomerase(CHI),chalcone synthase(CHS),flavanone 3-hydroxylase(F3H),dihydroflavonol 4-reductase(DFR)and leucoanthocyanidin dioxygenase(LDOX/ANS)and so on,which contains most of the key enzyme genes in the anthocyanin biosynthesis pathway.In addition,we identified one MYB gene(MYB114-like),two b HLH genes(b HLH33 and b HLHA-like)that may be associated with anthocyanin biosynthesis in rust-infected crabapple leaves by the method of constructing evolutionary tree and sequence amino acid sequence alignment.The results of RT-PCR showed that the up-regulated expression of these screened genes in the diseased tissues was consistent with the results of RNA sequencing.