| Rice sheath blight(SB)is one of the most serious diseases in rice worldwide,causing large loss to rice production.Rice resistance to R.solani belongs to quantitative genetic traits and is vulnerable to the environment.Rice germplasm with high resistance or immunity to SB has not yet been identified.In the previous studies in our laboratory,we inoculated YSBR1(relatively resistant to R.solani)and Lemont(susceptible to R.solani)with R.solani and analysised the DNA chip data,screening 109 differentially expressed genes.RNAi transgenic rice lines of a set of R.solani-responsive rice genes were generated by transformation using the rice variety TJ394.The RNAi transgenic lines were tested using the field inoculation method for rice adult plants in experimental field base.The RNAi lines of the candidate rice genes OsSBR1 showed resistance to R.solani.We evaluate the resistance level of rice cultivars by the detached-leaf method.At the same time,the subcellular localization,promoter expression analysis of the rice protein and comparative transcriptome analysis,were tested.The main results are as following:(1)RNAi transgenic rice lines,overexpression transgenic rice lines and knockout transgenic rice lines of resistance related gene OsSBR1 were generated by transformation.We performed qPCR assays of the SB pathogen in detached-leaf tissues and evaluating the disease level of rice cultivars.We have verified the qPCR results of disease severity by precise control of the conditions in inoculation experiments,and digital scanning of the disease lesions and statistical analysis of the relative lesion area.These results have verified the resistance phenotype of RNAi strains and knockout strains of OsSBR1 gene.And results have also verified the susceptible phenotype of overexpression strains of OsSBR1 gene.The above studies showed that OsSBR1 gene negatively regulated the resistance of rice sheath blight.(2)We built the instantaneous expression vector,which including all cDNA sequences of the pending genes.And we transformed the recombination plasmid into rice protoplasts.Images of fusion protein were then captured with the laser scanning confocal microscope.The results showed that the OsSBR1 is located in the cytoplasm.In addition,we detected the relative expression of OsSBRl in different tissues of rice,such as leaf,leaf sheath,young ear,seedling ear,young leaves,young sheath and young root,through qRT-PCR method.The results showed that the expression in leaf sheath was lower than other tissues of rice.At the same time,we also tested the GUS activity of OsSBR1 promoter-GUS transgenic rice tissue.The stain of leaf sheath is lighter,indicating that the OsSBRl gene has lower activity in leaf sheath tissue than other tissue of rice.(3)In order to study the molecular mechanism of resistance related gene OsSBR1,we performed transcriptome sequencing analysis.In this study,comparative transcriptome profiling of the OsSBR1-RNAi transgenic rice line and TJ394 with R.solani infection were investigated using RNA sequencing.Three biological replicates were performed for each genetype and each condition.With mapped to the rice genome sequence,it is found that genes with 69.03%-87.51%of their sequence covered by reads were generated in each library.In total,2455 differentially expressed genes(DEGs)were identified.GO functional analysis showed that most of the DEGs were involved in metabolic process,response to stimulus,catalytic activity,biosynthesis of secondary metabolites,plant-pathogen interaction,and planthormone signaling transduction pathways.This study enhances our understanding of the molecular mechanism of durable resistance to sheath blight,and provides candidate genes for rice germplasm with durable resistance to sheath blight,therefore has an important significance in both theoretical and practical aspects. |
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