Isolation and Characterization of NBS-LRR Resistance Gene Homology Sequences from Wheat

【Objective】In this study,resistance gene homology fragments from wheat were isolated using homology-based method.【Method】Two pairs of primers were designed according to the amino acid conserved regions NBS-LRR of the cloned plant disease resistance genes,and the reverse transcription-polymerase chain reaction(RT-PCR) was used.【Result】Three open-reading NBS resistance gene analogues(RGAs) have been obtained.They were named as PS13-1,PS13-2,and S2A2 respectively from the RNA of TcLr35 carrying the Lr35 gene conferring resistance against wheat leaf rust by a reverse transcription-polymerase chain reaction(RT-PCR).The nucleotide sequences of the three RGAs were 239bp,289bp and 539bp encoding 78,84 and 177 amino acids respectively.Homology research showed that the nucleotides of PS13-1 and PS13-2 were 91% identical to wheat powdery mildew resistance gene PM3b,and the nucleotide of S2A2 was 91% identical to the sequence from the mRNA of barley.BLASTp analysis indicated that three RGAs contained the conserved motifs of NB-ARC(such as P-loop,kinase2,kinase3a and transmembrance domain).Northern hybridization showed that the RGAs expression was not induced by Puccinia recondite inoculation suggesting that the gene be a constitutive gene with low abundance in wheat.【Conclusion】In this study,three resistance homology sequence were obtained in TcLr35,which provide the shortcut for the cloning of wheat leaf rust resistance genes.