| Apple(Malus domestica Borkh) is one of most important economic fruit crop in the world. And our country has the most widely cultivation area as well as the highest yield of apple in the world. The release of the apple genome provides important information to identify apple agronomic traits and resistance related genes at the genome level. The nonexpressor of pathogenesis related genes 1(NPR1) is the key regulator in systemic acquired resistance(SAR) signal pathways, which can be induced by salicylic acid or pathogens and control the expression of PR1 gene. In this study we identified all the members of NPR1 gene families from the apple genome by bioinformatics method. Then the multiple sequence alignment, phylogenetic analysis, exons–introns structure map were conducted to expound the evolutionary relationship and to assume the function of NPR1 family genes. Real-time fluorescent quantitative PCR was performed to study the apple NPR1 gene expression patterns in various tissues and organs. The expression difference of this family in susceptible cultivars and in resistant varieties were detected after inoculation of pathogens or SA,MeJA. The main results we obtained are as follows:1. Eight NPR1 homologous genes were identified from apple genome, and named MdNPR1- MdNPR8 respectively. These NPR1 genes products in apple were 490-590 amino acids in length, locating on seven different chromosomes.2. The eight NPR1 genes were cloned from two cultivars ‘Qinguan’ and ‘Pacific rose’. Four of theses genes MdNPR1, MdNPR2, MdNPR4, MdNPR5 show different forms of alternative splicing phenomenon. The sequence of MdNPR2 gene show polymorphism in the two varieties, but the other genes of this family have no sequence differences in two varieties.3. Sequence alignment of apple and Arabidopsis NPR1 genes show that apple NPR1 gene family contain conservative BTB/POZ, ankyrin repeat domain, were conservative at Cys82, Cys150, Cys155, Cys160, but only MdNPR1 are conservative at Cys216. Evolution analysis shows that apple and Arabidopsis were classified into 3 subgroups, but the number in every subgroup were significantly different(1 in group I, 5 in group II, 2 in group III). Gene structure analysis shows that apple and Arabidopsis have the same exons– introns structure.4. Apple NPR1 genes have different tissue-specific expression pattern. Among them, MdNPR1 expressed higher in leaf and pulp; the expression of MdNPR2, MdNPR3, MdNPR5 were highest in the pericarp; MdNPR4, MdNPR7, MdNPR8 expressed highest in flower, indicating the diversification of NPR1 gene expression pattern in different tissues and NPR1 gene family may have different functions in the different growth and development stages.5. The NPR1 gene expression in ‘Qinguan’ and ‘Pacific rose’ by exogenous SA, MeJA induction. In the two varieties, most NPR1 gene homologues were response to SA, MeJA, but the response time and extent of some genes have obvious differences in the two varieties.6. The NPR1 gene expression changes in ‘Pacific rose’ and M. baccata after inoculation of Marssonina coronaria. Among them, the expression of MdNPR1 in two varieties has contrary change trend at 6-48 h after inoculation; MdNPR2, MdNPR6 expressed significant differences in two varieties at 48 h after inoculation; MdNPR7-8 expressed show the same change trends after inoculation in two varieties. This implys that some of NPR1 genes may related to the different resistance of apple leaf spot. |
