Isolation of candidate disease resistance genes from enrichment library of Oryza minuta based on conserved domains
A strategy was described for the isolation of disease resistance genes from Oryza minuta by integrating the techniques of transformation-competent genomic library, RecA-mediated magnetic bead enrichment library and candidate disease resistance gene cloning. The principal advantages of this method were: simple, rapid and suitable. In this research, a transformation-competent genomic library with a volume of 2.68 × 105 clones was constructed for O. minuta; an enrichment library of disease resistance genes was further constructed with a volume of 4992 clones, from which 26 positive clones were screened by colony in situ hybridization. The end-clone sequencing of 13 representative positive clones showed that 6 clones were well matched with cloned disease resistance genes or located near the existing disease resistance genes. Full sequencing of a clone revealed a gene similar to a putative brassinosteroid LRR receptor kinase in japonica rice; the protein structure analysis suggested that it may be a disease resistance gene or functionally involved in a signal transduction pathway. These results indicate these clones may include new R genes and the strategy is feasible to clone new R genes from wild rice species.
Key words: Oryza minuta, disease resistance gene, transformation-competent genomic library, magnetic bead enrichment library, colony in situ hybridization, sequence analysis.