Constructing And Functional Analysis Of The Rice Candidated Disease Resistance Gene Library

Three strategies were employed for the cloning of rice R genes, including the use of the R genes enrichment library, the hpRNA meditated gene silencing mutant library and the domesticated wild rice genes, which provided a new method for plant gene cloning and enriched the cultivated rice R genes’ genetic diversity with resistance breeding materials.The wild rice species close to cultivated rice in the genome, gene expression and regulation are rich in R genes, and they are relatives. The usage of R genes from wild rice species for cultivated rice breeding is safer, more reliable and convenient than other species. The technology of the RecA protein meditated enrichment library construction was used for the screening of the wild rice DNA library. Two enrichment library for Dongxiang Common Wild Rice (O. rufipogon Griff.) with a volume of4608and Oryza minuta with a volume of16128were constructed. Further screening by colony in situ hybridization,118positive clones were isolated, including17for Dongxiang Common Wild Rice (O. rufipogon Griff.) and101for Oryza minuta. Comparing the restriction digestion in agoras gel,80positive clones without repeation were confirmed. The end-clone sequencing of80representative positive clones revealed that25clones can be located on the corresponding japonica rice genome, and51can not be located on the cultivated rice genome. Among the25candidate clones,15were perfectly matched with cloned japonica rice resistance genes or located near by resistance genes, indicating they were possible candidate resistance genes; Among the6whole genome sequenced clones,5have the conserved domains of R genes, suggesting that it may be an R gene. The candidate positive clones were transformed into the japonica rice varieties Taipei309, up till now a total of1938transgenic plants were obtained.758,1033and994transgenic rice materials were vaccinated for the resistance to rice bacterial leaf blight separately, but no resistance plants were identified.758transgenic materials were identified with the resistance to rice blast disease in the seedling stage under nature environment in Daweishan. All the plants were serious susceptible even death except43materials with moderate infections.1033transgenic materials were identified with the resistance to rice blast disease in the seedling stage under nature environment in Jishou, and all the plants were serious susceptible even death except19materials with moderate infections. The19materials with moderate infections and93T1transgenic materials were identified with the resistance to rice blast disease in the seedling stage under nature environment in Jishou, and all the plants were serious susceptible even death except6among19materials with moderate infections and9among93materials with moderate resistance.457transgenic rice materials were identified with the resistance to rice blast disease in the room with the strains of RB series; the material943has the resistance to strain RB19. 994transgenic rice materials were vaccinated for the resistance to rice bacterial leaf streak. The material341was identified with resistance to Xanthomonas oryzae pv. oryzicola strain RS105.HpRNA mediated mutant library has broad application prospects for avoiding the existing mutant libraries’ shortcomings. The hpRNA constructed by RMHR system was transformed into the japonica variety Taipei309for the construction of the mutant library and211transformations were obtained. The sequencing of36transformations revealed no disease resistance materials were found out. One lesion minic mutant R102was identified from the mutant library, sequence analysis revealed the function of this gene related to the plant nature defence.Many useful genes of wild species were lost in the evolution history, and the use of the domestication genes has benefits for the use of wild species germplasm. According to the sequence analysis of the domestication genes,2candidate clones were found out. By transforming them into the japonica variety Taipei309,86transformations were obtained. Four were identified with resistance to rice blast disease in the seedling stage under nature environment from43plants in Jishou.