Fine Mapping And Cloning Of Rice Bacterial Blight Resistance Gene Xa2

Bacterial blight(BB),caused by Xanthomonas oryzae pv.Oryzae,is one of the most serious disease of rice worldwide.Developing resistant cultivars is the most effective and economical means of controlling this disease.About thirty bacterial blight resistance genes in rice have been identified,six of them,Xa21,Xa1,Xa26,Xa27,xa5 and xa13 have been cloned successfully by a map based cloning approach.The BB resistance gene, Xa2,confers specific resistance to T7174(Japan Xoo race 2).It was identified and named by Sakaguchi(1967).Furthermore,Xa2 was mapped on the chromosome 4,linked to Xa1 with 2-16%recombination frequency.In this paper we report the result of fine mapping of the Xa2,and analysis the sequence structure,expression and function of the resistance gene analogs in the Xa2 region.1.Two F₂ populations are constructed to localize Xa2.The first population consisted of 1,101 F₂ plants derived from a cross between IR24 and IRBB2.The second population consisted of 2,769 F₂ plants derived from a cross between ZZA and IRBB2.In a primary analysis with 136 random F₂ plants of ZZA/IRBB2,it was found that Xa2 was located in approximately 20cM region.To accurately determine the locus of Xa2,120 SSR markers were used in this region.12 SSR markers were successfully used in genetic recombination analysis in IR24/IRBB2 population,while 20 in ZZA/IRBB2.The nearest SSR.markers to Xa2 are HZR970-6 and HZR645-2, which cover approximately 200kb region.2.Analysis the DNA sequence of Nipponbare chromosome 4 between markers HZR970-6 and HZR645-2,a BLASTX analysis tool,CCD v2.06 was used to predict protein structure and function.BLASTX analysis showed that only six predicted genes had sequence homology with known plant R genes.They all encoded NBS-LRR proteins and clustered together on Nipponbare BAC OSJNBb0085C12, The six predicted genes were designated RGA1,RGA5,GRA6,RGA7,RGA12 and RGA14.3.The expression of the six predicted genes was investigated by RT-PCR using total RNA from leaves of IR24 and IRBB2 at the booting stage inoculated with T7147. Member-specific primers,which flanking the predicted intron,were designed for each of predicted genes.RGA5 was expressed in IR24 and IRBB2,RGA12 was only expressed in the resistant parent IRBB2,but no expression of other predicted genes was detected.These results suggest that RGA5 and RGA12 were the best candidates for the Xa2 gene. 4.Individual RAGs as candidate Xa2 genes were amplified by LR-PCR,using genomic DNA of IRBB2 as a template.Two LR-PCR products were successfully amplified from IRBB2 and cloned into pMD 18-T vector,then recloned into the expression vector pCAMBIA1301S.The two candidate genes were transformed to TP309 and MDJ8,two susceptible rice materials,by Agrobacterium-mediated transformation. About 50-100 transformants of each gene were obtained.At the booting stage,all the transformants were inoculated with T7147 by the leaf-clipping method,but no plants showed resistance.5.A HELLSGATE vector was respectively used as a vector for cloning and expressing inverted repeat sequence corresponding to selected～500bp exon regions of RGA5 and RGA12.The RNAi constructs were transformed into IRBB2 by Agrobacterium-mediated transformation.