Isolation and Sequence Analysis of NBS Resistance Gene Analogues in Wild Rice

To explore resistance genes from wild rice,three sets of degenerate primers were designed based on the conservative domain of the nucleotide binding site(NBS) region from the cloned plant disease resistance genes(R) to isolate resistance gene analogues(RGAs) from genomic DNA of Oryza granulata,O.officinalis,O.alta,O.latifolia and O.punctata.After sequencing and analyzing by alignment,13 NBS RGAs were detected,11 of which had uninterrupted open reading frames(ORF) and contained the conserved motifs of NBS R genes such as P-loop,kinas-2,kinas-3a and GLPL,and the other two sequences were with immature stop codon.At the nucleotide level,the sequence identity of 12 RGAs ranged from 66% to 94% with the cloned NBS R genes from O.sativa,and between 67% and 84% with the cloned R genes from other plants.While the 12 RGAs deduced amino acid sequences showed identity with the cloned NBS type R gene from O.sativa ranging from 43% to 93%,and with the cloned R gene from other plants ranging from 37% to 79%.As for the other RGA,its nucleotide sequence identity with O.sativa putative NBS class R genes was 76%,and the deduced amino acid sequence identity was 74%.To further analyze the expression of these RGAs,RT-PCR was preformed.The results showed that RN1BD5,RN1BD10,RN1GG2 and RN1YY6 could express,which indicated that these fragments might be partial sequence of functional R genes;whereas RN1KY9 and RN1GG5 didn′t express,which indicated they might be residual arti