Liu J-J, Ekramoddoullah A K M
Pacific Forestry Centre, Canadian Forest Service, Natural Resources Canada, 506 West Burnside Road, V8Z 1M5, Victoria, British Columbia, Canada.
Mol Genet Genomics. 2003 Dec;270(5):432-41. doi: 10.1007/s00438-003-0940-1. Epub 2003 Oct 28.
Western white pine ( Pinus monticola Dougl. ex. D. Don., WWP) shows genetic variation in disease resistance to white pine blister rust ( Cronartium ribicola). Most plant disease resistance (R) genes encode proteins that belong to a superfamily with nucleotide-binding site domains (NBS) and C-terminal leucine-rich repeats (LRR). In this work a PCR strategy was used to clone R gene analogs (RGAs) from WWP using oligonucleotide primers based on the conserved sequence motifs in the NBS domain of angiosperm NBS-LRR genes. Sixty-seven NBS sequences were cloned from disease-resistant trees. BLAST searches in GenBank revealed that they shared significant identity to well-characterized R genes from angiosperms, including L and M genes from flax, the tobacco N gene and the soybean gene LM6. Sequence alignments revealed that the RGAs from WWP contained the conserved motifs identified in angiosperm NBS domains, especially those motifs specific for TIR-NBS-LRR proteins. Phylogenic analysis of plant R genes and RGAs indicated that all cloned WWP RGAs can be grouped into one major branch together with well-known R proteins carrying a TIR domain, suggesting they belong to the subfamily of TIR-NBS-LRR genes. In one phylogenic tree, WWP RGAs were further subdivided into fourteen clusters with an amino acid sequence identity threshold of 75%. cDNA cloning and RT-PCR analysis with gene-specific primers demonstrated that members of 10 of the 14 RGA classes were expressed in foliage tissues, suggesting that a large and diverse NBS-LRR gene family may be functional in conifers. These results provide evidence for the hypothesis that conifer RGAs share a common origin with R genes from angiosperms, and some of them may play important roles in defense mechanisms that confer disease resistance in western white pine. Ratios of non-synonymous to synonymous nucleotide substitutions (Ka/Ks) in the WWP NBS domains were greater than 1 or close to 1, indicating that diversifying selection and/or neutral selection operate on the NBS domains of the WWP RGA family.
西方白松（Pinus monticola Dougl. ex. D. Don.，WWP）在对白松疱锈病（Cronartium ribicola）的抗病性方面表现出遗传变异。大多数植物抗病（R）基因编码的蛋白质属于一个超家族，该超家族具有核苷酸结合位点结构域（NBS）和C端富含亮氨酸重复序列（LRR）。在这项研究中，采用PCR策略，基于被子植物NBS-LRR基因NBS结构域中的保守序列基序，使用寡核苷酸引物从西方白松中克隆抗病基因类似物（RGA）。从抗病树中克隆出67个NBS序列。在GenBank中进行BLAST搜索发现，它们与被子植物中已明确特征的R基因具有显著的同源性，包括亚麻的L和M基因、烟草的N基因以及大豆的LM6基因。序列比对表明，西方白松的RGA包含在被子植物NBS结构域中鉴定出的保守基序，特别是那些TIR-NBS-LRR蛋白特有的基序。对植物R基因和RGA的系统发育分析表明，所有克隆的西方白松RGA可以与携带TIR结构域的知名R蛋白一起归入一个主要分支，这表明它们属于TIR-NBS-LRR基因亚家族。在一个系统发育树中，西方白松RGA进一步细分为14个簇，氨基酸序列同一性阈值为75%。用基因特异性引物进行cDNA克隆和RT-PCR分析表明，14个RGA类别中的10个类别在叶片组织中表达，这表明一个庞大且多样的NBS-LRR基因家族可能在针叶树中发挥功能。这些结果为针叶树RGA与被子植物的R基因有共同起源这一假说提供了证据，并且其中一些可能在赋予西方白松抗病性的防御机制中发挥重要作用。西方白松NBS结构域中非同义核苷酸替换与同义核苷酸替换的比率（Ka/Ks）大于1或接近1，表明多样化选择和/或中性选择作用于西方白松RGA家族的NBS结构域。
