Shen K A, Meyers B C, Islam-Faridi M N, Chin D B, Stelly D M, Michelmore R W
Department of Vegetable Crops, University of California, Davis 95616, USA.
Mol Plant Microbe Interact. 1998 Aug;11(8):815-23. doi: 10.1094/MPMI.1998.11.8.815.
The recent cloning of genes for resistance against diverse pathogens from a variety of plants has revealed that many share conserved sequence motifs. This provides the possibility of isolating numerous additional resistance genes by polymerase chain reaction (PCR) with degenerate oligonucleotide primers. We amplified resistance gene candidates (RGCs) from lettuce with multiple combinations of primers with low degeneracy designed from motifs in the nucleotide binding sites (NBSs) of RPS2 of Arabidopsis thaliana and N of tobacco. Genomic DNA, cDNA, and bacterial artificial chromosome (BAC) clones were successfully used as templates. Four families of sequences were identified that had the same similarity to each other as to resistance genes from other species. The relationship of the amplified products to resistance genes was evaluated by several sequence and genetic criteria. The amplified products contained open reading frames with additional sequences characteristic of NBSs. Hybridization of RGCs to genomic DNA and to BAC clones revealed large numbers of related sequences. Genetic analysis demonstrated the existence of clustered multigene families for each of the four RGC sequences. This parallels classical genetic data on clustering of disease resistance genes. Two of the four families mapped to known clusters of resistance genes; these two families were therefore studied in greater detail. Additional evidence that these RGCs could be resistance genes was gained by the identification of leucine-rich repeat (LRR) regions in sequences adjoining the NBS similar to those in RPM1 and RPS2 of A. thaliana. Fluorescent in situ hybridization confirmed the clustered genomic distribution of these sequences. The use of PCR with degenerate oligonucleotide primers is therefore an efficient method to identify numerous RGCs in plants.
最近从多种植物中克隆出了抗多种病原体的基因，这表明许多基因具有保守的序列基序。这为通过聚合酶链反应（PCR）使用简并寡核苷酸引物分离大量其他抗性基因提供了可能性。我们用从拟南芥RPS2和烟草N的核苷酸结合位点（NBS）基序设计的低简并度引物的多种组合，从生菜中扩增抗性基因候选物（RGC）。基因组DNA、cDNA和细菌人工染色体（BAC）克隆成功用作模板。鉴定出四个序列家族，它们彼此之间的相似性与其他物种的抗性基因相同。通过几个序列和遗传标准评估扩增产物与抗性基因的关系。扩增产物包含具有NBS特征性附加序列的开放阅读框。RGC与基因组DNA和BAC克隆的杂交揭示了大量相关序列。遗传分析表明四个RGC序列中的每一个都存在成簇的多基因家族。这与抗病基因成簇的经典遗传数据相似。四个家族中的两个定位于已知的抗性基因簇；因此对这两个家族进行了更详细的研究。通过在与NBS相邻的序列中鉴定出与拟南芥RPM1和RPS2中相似的富含亮氨酸重复（LRR）区域，获得了这些RGC可能是抗性基因的额外证据。荧光原位杂交证实了这些序列的成簇基因组分布。因此，使用简并寡核苷酸引物进行PCR是鉴定植物中大量RGC的有效方法。
