NPR1-like genes from cDNA of rosaceous trees: cloning strategy and genetic variation

In this work, we report the isolation and characterization of NPR1-like gene fragments from nine species belonging to the rosaceous subfamilies Pomoideae and Amygdaloideae. Seventy-three NPR1-like gene fragments with uninterrupted open reading frame (ORF) were isolated from the expressed mRNA through a reverse transcriptase PCR-based strategy, most of them ranging from 511 to 827 bp. The deduced amino acid sequences of these gene fragments shared structural features with known NPR1(-like) proteins: ankyrin repeat and the broad complex tramtrack and bric-a-brac/pox virus and zinc finger domain. The highest homology values were found with the NPR1-like gene from Lycopersicon esculentum and genes within the NPR4 phylogenetic group from Arabidopsis thaliana, suggesting that they are the NPR4-like orthologs. Phylogenetic analysis confirmed this conclusion and demonstrated the complexity of the gene families. The ankyrin domain was analyzed in detail and an ankyrin consensus was determined. A comparison of this consensus with a statistically derived ankyrin repeat, as reported by other authors, enabled the prediction of the ankyrin repeat secondary structural elements. In addition, a comparison with some important structural and functional features of NPR1 was performed.