Gene Expression Analysis Of Endogenous Growth Of Sclerotinia Sclerotiorum Strain DT-8 In Brassica Napus

Sclerotinia sclerotiorum(Lib.)de Bary,which has a wide range of host and geographical distribution,is one of the most devastating plant pathogenic fungus in the world.The S.sclerotiorumn strain DT-8 infected by the Sclerotinia sclerotiorum hypovirulence-associated DNA covirus 1(SsHADV-1)and was hypovirulent.Based on the previous research,Brassica napus seeds could germinate and grow normally after treated with the mycelium of DT-8.In particular,DT-8 and SsHADV-1 could be detected in the tissues of B.napus,suggesting that after SsHADV-1 infected,the necrotrophic plant pathogen S.sclerotiorumn could be transformed into endophytic fungi with no significant adverse influence on host plant.Furthermore,endogenous growth of DT-8could improve the resistance of B.napus to S.sclerotiorum.Therefore,DT-8 has a high potential for biocontrol and further research.In this study,the feasibility of R-PCR was verified at single gene level,genome level and cDNA level of rape with endogenous DT-8.After the R-PCR method was optimized,the main enriched pathway of up-regulated genes in rape and expressed genes in S.sclerotiorum were analyzed.The main results were as followed:1.At the level of single gene,genome and cDNA,we explored the preparation system of R-PCR primer and template and the concentration ratio of the primer and template at the removing stage.At the level of single gene,excellent elimination effect was obtained when the concentration ratio of primer and template was 4:1～8:1.At the genomic level,due to the fragments of T-A cloning were too short,the adapter sequences were obtained.At the cDNA level,the gene expressed in rape and DT-8 can be cloned simultaneously,indicating that R-PCR can be used for the analysis of differentially expressed genes in this condition.2.The R-PCR technique was optimized.The proportion of S.sclerotiorum gene in the eliminate products could be maximized by using rape epicotyl as sample and increasing the cycle number to 16 at the first stage.3.The R-PCR eliminate products were sequenced by high-throughput sequencing,and the Gene Ontology Function and Pathway Enrichment was analyzed.Gene expression data showed that resistance-related and photosynthesis-related pathway were activated in rapeseed plants after DT-8 endogenous grown.For DT-8,it maintained an endophytic state mainly by regulating the substance and energy metabolism and cell proliferation.In this research,R-PCR was successfully applied to the differential expression analysis DT-8 and rape interaction.Combining with high-throughput sequencing,lots of candidate genes involved in the interaction between B.napus and S.sclerotiorum were screened.Our results provide a new research method and idea for the further study of the transformation of necrotrophic plant pathogenic fungi to endophytic fungi and the interaction mechanism between endophytic fungi and host plants.