芸香科柑橘黄龙病较耐病寄主植物NBS-LRR类RGA的分离鉴定及表达分析

itrus production is seriously affected by HLB to which no resistant plant is available in the cultivated germplasm. Degenerate primers designed based on conserved NBS (nucleotide binding site) motifs contained in some plant resistance genes were used to isolate analogous sequences called resistance gene analogs (RGAs). About xx resistance gene analogs (RGAs) were cloned from several spices related to HLB using PCR method with primers above-mentioned. The BLASTN searches revealed that 43 DNA fragments showed different sequence homology with known plant R-genes or deposited RGAs and 12 sequences could be translated to polypeptides without stop codons. These RGAs and 12 deduced amino acid were analyzed by Clustalx and DNAMAN software. Sequence analysis showed these RGAs contain the conserved domains P-loop, Kinase-2a, Kinase-3a, and HD, which was conserved in NBS-LRR type disease resistance gene. These 12 RGAs shared 37.39%-43.43% identity with the resistance genes of TobaccoN、FlaxL6、Arabidopsis thaliana RPS2、RPS5、RPP8、RPM1. Gene duplication followed by sequence divergence were proposed as the mode for the evolution of a large number of distantly or closely related RGA genes in plants, and this mode may play a role in the generation of new resistance speci?city and cloning related resistance genes about HLB. In addition, using PCR amplification from cDNA of Citrus maxima and RFLP, 5RGAs were successfully obtained, with GenBank accession numbers of HM777043-HM777043. The Quantitative Real-time PCR results showed that expression of 5 RGAs changed differently. Therefore, it suggested that these RGAs might play important roles in disease resistance in defense response pathway.