Isolation and Characterization of NBS-LRR Resistance Gene Analogs from Sugarcane

For the purpose of isolating resistance gene analogs (RGAs) from sugarcane (Saccharum officinarum Roxb.) with primers from the conservative sequences of nucleotide-binding site (NBS), 6 forward and 10 backward primers were designed according to the conserved motifs in the NBS regions of 3 typical NBS-LRR resistance genes RPS2, N, and L6. The homologous PCR was used to amplify NBS sequences from genomic DNA and cDNA of smut-resistant sugarcane variety NCo376. A total of 11 RGAs were obtained, of which 5 from the genomic DNA and 6 from the cDNA. Sequence analysis showed that these RGAs comprised of the conserved domains P-loop, Kinase-2a, Kinase-3a, and HD, which was conserved in NBS-LRR type of disease-resistance gene. The last amino acid in the alignment was residue W in LLVLDDV (W/D) motif, which is typical in non-TIR-NBS-LRR type of genes, indicating only non-TIR-NBS-LRR type resistance genes in the genome of sugarcane. The 11 RGAs, together with RPS2 and Xa1, were clustered into one group, and N and L6 were in another group. One RGA, termed PIC (EF059974), was validated through real-time PCR. The result showed that the expression of PIC gene was induced by Ustilago scitaminea and salicylic acid, but inhibited by hydrogen peroxide. The PIC gene had constitutive expressions in leaves, stalks, and roots of sugarcane, with the strongest expression in leaves.