2009•ACTA AGRONOMICA SINICARequires access
Isolation and Characterization of NBS-LRR Resistance Gene Analogs from Sugarcane
Que You
Open publisher page 0 citations
Abstract
The large group of plant disease resistance (R) genes that share similar structures possesses a predicted nucleotide-binding site (NBS) domain. NBS domains of this class of R genes show highly conserved amino acid motifs, which makes it possible to isolate resistance gene analogs (RGAs) by PCR with degenerate primers. According to the conserved motifs in the NBS regions of the three typical NBS-LRR type resistance genes (RPS2, N, and L6), five degenerate and one non-degenerate primers were designed to correspond to P-loop motif in sense direction, and nine degenerate plus one non-degenerate primers were made corresponding to the HD motif in the anti-sense direction. Then, the homologous PCR was used to amplify NBS sequences from genomic DNA and cDNA using sugarcane variety NCo376 with smut resistance. In all, eleven RGAs were obtained, five from DNA (EF059973, EF059974, EF059975, EF059976, and EF059977) and six from cDNA (EF155648, EF155649, EF155650, EF155651, EF155652, and EF155653). Sequence analysis showed that RGAs comprised the conserved domains P-loop, Kinase-2a, Kinase-3a, and HD, which was conserved in NBS-LRR type disease resistance gene. Cluster analysis showed that eleven RGAs and RPS2 and XA1 were clustered into one group, and N and L6 were divided into another group. Further, amino acid sequences showed that their last amino acid in alignment was residue W in LLVLDDV(W/D) motif, which is typical to non-TIR-NBS-LRR type gene. It was suggested that only non-TIR-NBS-LRR but not TIR-NBS-LRR type resistance genes ex- isted in sugarcane genome. One RGA termed PIC (EF059974) was randomly selected for function validation through Real-time PCR. The result showed that expression of PIC gene could to some extent be influenced by U. scitaminea, SA and H2O2, and had the characteristics of constitutive expression and tissue-specific. The RGA cloned in this experiment may provide the shortcut for cloning of sugarcane disease resistance gene.