烟草表达抗病基因同源物(RGA)的鉴定及RGA-SSR标记的开发

Identification of Expressed Resistance Gene Analogues(RGAs) and Development of RGA-SSR Markers in Nicotiana

烟草是研究植物与病原菌互作的理想材料。鉴定烟草抗病基因及其同源物对揭示抗病机制具有重要意义。近年来公共数据库不断增长的EST序列为烟草表达RGA的鉴定提供丰富的数据。本研究通过拼接GenBank收录的412325条烟草EST序列，获得149606条Uni．EST序列。随后利用已克隆的112个植物R基因蛋白序列对其扫描，检测出1113个NtRGA，其中有273、546、53、102和30个分别包含NBS-LRR、LRR．PK、LRR、PK和Mlo结构域，另有109个未检测到结构域。通过序列比对将1071个NtRGA定位于Ⅳ．benthamiana基因组712个位点上。经搜索，从72个NtRGA中检测出78个SSR，根据其侧翼序列设计64对引物。54对成功从烟草基因组DNA中扩增出清晰条带，9对在24个普通烟草品种间检测出多态性，检出等位基因数2～4个，平均2．56个；41对在6个烟草种间检测出多态性，检出等位基因数2～4个，平均2．61个。

Tobacco is an ideal experimental system for studing on plant-pathogen interaction. Identification of tobacco R gene and resistance gene analogs is propitious to elucidating the underlying resistant mechanisms. In recent years, the growing public to- bacco EST data provide rich source for identifying expressed RGA. In this study, 149 606 Uni-EST were assembled from 412 325 ESTs of tobacco in GenBank. By scanning the Uni-EST with 112 plant R gene protein sequences 1113 NtRGAs were identified. These expressed RGAs comprised 273, 546, 53, 102, and 30 of NBS-LRR, LRR-PK, LRR, PK, and Mlo domains encoding R genes, respectively. No domain was detected in the rest of 109 RGAs. By aligning sequence 1079 NtRGAs were allocated on 712 loci in N. benthamiana genome. A total of 78 simple sequence repeats （SSRs） were identified from 72 NtRGAs. Sixty-four primer pairs were designed base on the flanking sequence of SSR. Among them, 54 primer pairs were amplified with clear bands from tobacco genomic DNA. Nine primer pairs were detected to have polymorphism among 24 varieties of Nicotiana tabacum with two to four alleles （on average 2.56 alleles）. Forty-one primer pairs were detected to have polymorphism among six species in Nicotiana with two to four alleles （on average 2.61 alleles）.