水稻中一个NBS-LRR抗病同源基因家族的克隆和分析

利用克隆的抗病基因同源序列RS13作为探针，从水稻IR64的BAC文库中筛选到4个阳性克隆，其中一个克隆14E19能够覆盖其余3个克隆。对14E19进行全序列测定和分析，获得了73kb的全长DNA序列，基因预测显示其上有4个编码NBS-LRR结构域的基因(NL)，分别命名为NL-A，B，C和D。对具有相同基因组背景的IRBB56同一染色体位置上跨度更大的BAC克隆106P13进行分析，发现其上有10个NL同源拷贝，其中4个同14E19上的NL一样。搜索日本晴、93—11、广陆矮4号的序列，发现三者有类似的同源序列。但与已知的NBS-LRR抗病基因同源性较低，说明NL是一个至少由10个成员(分别命名为NL-A至J)组成的新基因家族。对NL家族进行RT-PCR和cDNA库筛选分析，发现NL-B基因能够在抗白叶枯病品系IRBB4中表达，暗示该基因参与了抗病反应。

Cloning and Analysis of a New NBS-LRR Resistance Gene Family in Rice

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.