Wang Shi-Quan, Zhang De-Chun, Li Ping, Wang Xu-Dong, Li Shi-Gui, Zhu Li-Huang, Zhai Wen-Xue
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
Yi Chuan Xue Bao. 2005 Jul;32(7):704-11.
Sequence-based gene isolation has been a practical approach for plant resistance gene cloning. In this study, RS13, a cloned rice sequence with the NBS (nucleotide-binding site) domain of resistance genes, was used as a probe to screen a bacterial artificial chromosome (BAC) library of rice variety IR64,and four positive clones were obtained. Of them the clone 14E19 covered the other three clones and was sequenced through a shotgun approach. The whole sequence of the insert fragment of 14E19 was assembled into approximately 73 kb in length. Genes on the whole assembled sequence were predicted,and four genes encoding NBS and LRR (leucine-rich repeat) domains were found, named as NL-A, B, C and D respectively. For further analysis, another longer BAC clone,106P13, covering 14E19 on the same chromosome position was identified from a BAC library of IRBB56 which had the same genome background with IR64. Ten NL-homologous copies were discovered on the sequence of the BAC clone 106P13, and four copies were identical with those on 14E19. The similar homologous sequences were also found in the genomic sequences of Nipponbare,93-11 and Guangluai4. However, NL sequences were less homologous with the known NBS-LRR resistance genes. This result indicated that NL was a new NBS-LRR gene family and was composed of ten members at least. RT-PCR and cDNA screening displayed that NL-B expressed in a bacterial blight-resistant rice variety IRBB4, indicating the gene was possibly involved in resistance reactions.
基于序列的基因分离一直是植物抗性基因克隆的一种实用方法。在本研究中，RS13是一个已克隆的具有抗性基因NBS（核苷酸结合位点）结构域的水稻序列，用作探针筛选水稻品种IR64的细菌人工染色体（BAC）文库，获得了4个阳性克隆。其中克隆14E19覆盖了其他3个克隆，并通过鸟枪法进行测序。14E19插入片段的全序列组装后长度约为73 kb。对整个组装序列上的基因进行预测，发现4个编码NBS和LRR（富含亮氨酸重复序列）结构域的基因，分别命名为NL-A、B、C和D。为进一步分析，从与IR64具有相同基因组背景的IRBB56的BAC文库中鉴定出另一个更长的BAC克隆106P13，其在同一染色体位置覆盖14E19。在BAC克隆106P13的序列上发现了10个NL同源拷贝，其中4个与14E19上的相同。在日本晴、93-11和广陆矮4的基因组序列中也发现了相似的同源序列。然而，NL序列与已知的NBS-LRR抗性基因的同源性较低。这一结果表明NL是一个新的NBS-LRR基因家族，至少由10个成员组成。RT-PCR和cDNA筛选显示NL-B在抗白叶枯病水稻品种IRBB4中表达，表明该基因可能参与抗性反应。
