Molecular Mechanism Analysis Of Resistance Or Susceptibility To Sclerotinia Sclerotiorum Of Brassica Napus Through RNA-Seq

Stem rot caused by Sclerotinia sclerotiorum is a worldwide disease, which is also one of the first three diseases of oilseed rape, seriously affecting the yield and quality of rape. The pathogen S. sclerotiorum is a kind of necrotrophic fungus with wide host range, and the pathogenic mechanism of which is complex. We have not yet found the antigen and the effective prevention and control methods to protect plant against this pathogen.In this study, we analyzed the transcription information of xiangyou15and98C40NL inoculated with S. sclerotiorum at three time point, which were before inoculated,12hours and24hours after inoculation, through RNA-Seq, in order to realize the dynamic changes of gene transcription of brassica napus under the stress of Sclerotinia sclerotiorum. We also studied the effect of secondary metabolites against S1. sclerotiorum, including alkaloids and glucosinolate. According to the results, we analyzed the signal transduction pathways and secondary metabolisms involved in the interaction between Basic napes and S. sclerotiorum. We also cloned calcium dependent protein kinase gene BnCDPK1in Brassica napus and analyzed its transcription characteristics between different organisms and with the stress of S. sclerotiorum. These studies laid the foundation for further study on the molecular mechanism of resistance to S. sclerotiorum.Main results are as follows:1. Six pools of mRNA samples, two Brassica napus cultivars Xiangyou15and98C40*xiangyoul5at three time points inoculated with S. sclerotiorum which were before inoculated,12hpi and24hpi, were used to build libraries for RNA-Seq. De Novo assemblies yielded169343unigenes with an average length of826bp.2. To identify the putative function of the unigenes, we performed BlastX alignments against NCBI non-redundant databases NR, Swiss-Prot, COG, and KEGG databases. There were88412unigenes matched to one or more database. According to the NR annotation, there were49378unigenes mapped to different function terms of GO database. A total of49276unigenes were classified to25categories according to their sequence homology to COG database.22785unigenes mapped to KEGG database, which participate in301pathways. The number of unigenes involved in the metabolism pathway is most which was 22390. Besides, there were806unigenes participating in the plant hormone signal transduction, and766unigenes involved in plant-pathogen interaction pathway respectively.3.87803CDS were forecasted according to the sequence alignment to protein databases, and52416CDS were deduced through ESTScan analyses. The lengths of25215CDS were above1000bp, which occupied18%of all the CDS.4. We analyzed the transcription differences between different time point of both high susceptible and high resistant cultivar, and the same time point between different cultivars. There were31532genes differentially expressed above2times in susceptible cultivar after inoculated with S. sclerotiorum compared to before inoculated, in which,2702genes of which were steadily up-regulated from12hpi to24hpi, and1611genes continues to down regulated, while27219exhibited complex changes. The number of differentially expressed genes in high resistant cultivar was3168,3204and3213of which were continuously up-regulated and down-regulated respectively, the rest25371genes showed variation at different time points. There were also many genes with different transcription abundance between the two cultivars.19378genes in all exhibit expression differences,110of which were up-regulated in all the three time points and60were down-regulated.5. PAMPs triggered immunity (PTI) is very important for Brassica napus defend to S. sclerotiorum. Pattern recognition receptors (PRRs) mediated PTI and help to resist necrotrophs. We found that the transcription abundance of PRRs related genes include RLKs, CRPKs and LRPKs were up regulated after inoculated, and have significant differences between the two cultivars.6. Plant hormone mediated the signal transduction response to the stress of S. sclerotiorum. The genes related to the biosynthesis and signal transduction of ethylene, JAs and ABA showed expression differences which were elevated after treated with S. sclerotiorum and the expression level was higher in resistant cultivar compared with the susceptible one. We also found that the synthesis of jasmonic acid and linolenic acid in the tissue were induced by S. sclerotiorum. It implies that JAs, ET and ABA signal transductions triggered the defense responses.7. The signal transduction pathway mediated by Ca2+was involved in the resistance reaction of Brassica napus. Ca+receptor proteins and protein kinases, such as CDPKs, CMLs. CBLs and CIPKs were up-regulated by S. sclerotiorum, and the transcription abundance of which was higher in the high resistant cultivar.8. Secondary metabolisms are important material for resisting against S. sclerotiorum infection. We found that the genes related to the biosynthesis and metabolism of alkaloid and glucosinolate expressed differently and put effect on preventing infection of S. sclerotiorum.