Cloning And Functional Analysis Of A Rice Blast Resistance Gene BRG8

Rice blast is a kind of fungal disease that impact rice yield and quality for a long time.Over the world,about eitghty contries and districts could suffer from blast disease.Cloning resistance gene and exploring the mechanism is of great value for rice breeding improvement.At present,the main approach to isolate resistance gene is by using bi-parient population,via map-based cloning method.This study proceeded candidate gene isolation based on a previous genome-wide association study.As a result,we successfully cloned a rice blast resistance gene,and performed functional analysis via molecular biology method.The main results are presented as follow:1.Via genome-wide association mapping,this study cloned a blast resistance gene located on position 5.2 Mb of rice chromosome 8,designated as BRG8.The transgenetic experiments confirmed blast resistance of BRG8.Moreover,BRG8 is able to enhance rice resistance to Xanthomonas oryzae.2.Expression pattern analysis showed BRG8 was mainly expressed in rice leaf but not in other tissues.RNA signal of BRG8 could be detected in each growth stage,while the signal was much more significant in tillering stage.BRG8 is not induced by Magnaporthe oryzae.Subcellular localization experiment indicated BRG8 protein was mainly located in cytoplasm.3.Analysis on pathogensis-related genes demonstrated BRG8 enhanced expression of two PR genes,PAL,PBZ1.Genes involved in resistance signal transduction like,WRKY45,WRKY53,are also enhanced by BRG8.RNA-seq analysis using BRG8 transgenetic plants and control plants detected 136 differential expressed genes.Except several PR genes,genes encoding cellulose/ligin synthesis enzymes,glycosyl transferase,GDSL-like lipase/acylhydrolase and Fasciclin domain containing protein were also detected as increasing expression level.These results revealed a wide regulation medidated by BRG8.Moreover,the content of cellulose and ligin were measured to verified gene expression level suggested their role in blast resistance.4.With reported genomic data,we performed a comprehensive analysis on genetic variation of BRG8.Sixteen haplotypes were identified in cultivated rice,while five of them were rare types.Further analysis on the phylogenesis with sequence from several wild rice species revealed a clear evolution relationship of BRG8.Among genus oryza with AA genomes,two major classes were found.The first group contains O.barthii,O.nivara,O.glaberrima,most indica types,tropical japonica,and Aus type.The second group includes O.meridionalis,O.ruffipogon,O.glumipatula,temperate japonica and few indica cultivars.The resistant haplotype,V(Hap3),has a close genetic distance with O.ruffipogon suggests this type may be infiltrated from wild rice.