晚熟温州蜜柑 NBS-LRR类抗病基因同源序列的克隆及分析

摘要

为加深对柑橘黄龙病抗性机理的了解和为黄龙病抗性基因的克隆提供依据，根据已知植物抗病基因NBS-LRR保守结构域设计简并引物，以柑橘黄龙病抗性品种“晚熟温州蜜柑”为供试材料，对其基因组DNA进行PCR扩增、克隆和序列测定，结果共获得17条抗病基因同源序列（resistance gene analogs，RGAs），其在NCBI中的登录号为KJ019189～KJ019199，KR815564～KR815569。序列分析表明，这17个RGAs与甜橙、柚等柑橘属中推测的一些抗病蛋白基因或其他相关蛋白基因具有显著高的同源性。其中一些RGAs与拟南芥抗霜霉病RPP13基因、柑橘抗衰退病毒基因Ctv或烟草抗TMV基因N具有51．7％～79．0％的氨基酸序列同源性。依据论文结果，笔者认为，晚熟温州蜜柑中存在较丰富的NBS-LRR类抗病基因，但具体哪些病基因与抗黄龙病相关尚需进一步研究。%In order to deepen understanding of the resistant mechanism of Citrus to Candiadatus Liberi-bacter asiaticus( CLas) and lay the foundation for cloning of citrus huanglongbing( HLB)-resistant gene,a pair of degenerate primers was designed from the conserved domains of NBS-LRR type plant disease-resistant gene and used for amplifying the disease-resistant gene analogs( RGAs) by polymerase chain reaction( PCR) from the genomic DNA of Citrus unshiu Marc.,a citrus cultivar resistant to HLB.After cloning and sequencing the PCR products,the nucleotide sequences and their deduced amino acid sequences of 17 RGAs were obtained and deposited in GenBank with the accession number of KJ019189-KJ019199 and KR815564-KR815569.Se-quence analysis showed that these 17 RGAs shared significantly high homology with some putative disease-re-sistant genes and other protein genes in Citrus sinensis or Citrus clementina, some of which shared 51.7%-79.0%amino acid sequence homology with the known disease gene of RPP13,Ctv,or N.Based on these re-sults,it is concluded that fairly rich NBS-LRR type plant disease-resistant genes exist in Citrus unshiu Marc. However,further research is needed to clarify which resistant genes are related with citrus huanglongbing.