Isolation and characterization of resistance gene homology sequence from wheat

Two pairs of primers were designed according to the amino acid conserved regions of reported plant disease resistance gene which encode proteins that contain nucleotide-binding site (NBS) and leucine-rich repeats (LRR). Two resistance gene analogues (RGAs) had been obtained, which were named as S2A2 and LRR1, from the RNA of TcLr35 containing Lr35 gene conferring resistance against wheat leaf rust by reverse transcription polymerase chain reaction (RT-PCR). S2A2 had 539 bp encoding 177 amino acids and LRR1 had 215 bp encoding 58 amino acids. Homology search in GenBank showed that S2A2 was 91% identical to a resistance gene homology EST sequence of barley. BLASTp analysis showed that S2A2 contained the conserved motifs of NB-ARC: P-loop, kinase 2, kinase 3a and transmembrance domain and the amino acid of LRR1 was 74% identical to Xa21. Northern hybridization showed that these RGAs expression was not induced by Puccinia triticina inoculation, indicating that they are constitutive genes with low abundance in wheat. 3'RACE-PCR was carried out with gene-specific primers and the 3'-universal primer provided in the kit. An amplified fragment of 567 bp that overlapped with the LRR1 sequence by 184 bp was obtained. A 598 bp fragment was determined to be the sequence of LRR1 except the 5'cDNA end encoding a predicted polypeptide of 153 amino acid containing three LRR repeats. The fragment also contained 84 bp untranslated region (UTR) at 3'-ends and 21 bp poly(A) tail. The study paved the way for the cloning of Lr35 or the resistance-related gene.