The broad-spectrum rice blast resistance gene Pi9 was cloned using a map-based cloning strategy. Sequencing of a 76-kb bacterial artificial chromosome (BAC) contig spanning the Pi9 locus led to identification of six tandemly arranged resistance-like genes with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) (Nbs1-Pi9–Nbs6-Pi9). Analysis of selected Pi9 deletion mutants and transformation of a 45-kb fragment from the BAC contig into the susceptible rice cultivar TP309 narrowed down Pi9 to the candidate genes Nbs2-Pi9 and Nbs3-Pi9. Disease evaluation of the transgenic lines carrying the individual candidate genes confirmed that Nbs2-Pi9 is the Pi9 gene. Sequence comparison analysis revealed that the six paralogs at the Pi9 locus belong to four classes and gene duplication might be one of the major evolutionary forces contributing to the formation of the NBS–LRR gene cluster. Semiquantitative reverse transcriptase (RT)–PCR analysis showed that Pi9 was constitutively expressed in the Pi9-resistant plants and was not induced by blast infection. The cloned Pi9 gene provides a starting point to elucidate the molecular basis of the broad-spectrum disease resistance and the evolutionary mechanisms of blast resistance gene clusters in rice.
通過圖位克隆的方法克隆了一個水稻稻瘟病廣譜抗性基因Pi9。對包含Pi9位點的長76 kb的細(xì)菌人工染色體重疊群進(jìn)行測序，鑒定出了6個串聯(lián)排列的含有核苷酸結(jié)合位點(NBS)及富含亮氨酸重復(fù)(LRR)的抗病相關(guān)基因(Nbs1-Pi9–Nbs6-Pi9)。對Pi9選擇性缺失突變體的分析以及將45kb的BAC重疊群轉(zhuǎn)到感病品種TP309，將Pi9的候選基因縮小為Nbs2-Pi9和Nbs3-Pi9。對轉(zhuǎn)化有單個候選基因的轉(zhuǎn)基因植株進(jìn)行抗病檢測確定了Nbs2-Pi9為Pi9基因。序列比較分析表明位于Pi9位點的這6個旁系同源基因?qū)儆?個基因家族，同時基因復(fù)制可能是這個NBS–LRR基因簇形成的主要進(jìn)化選擇壓力之一。半定量RT-PCR分析表明Pi9在抗病植株中持續(xù)表達(dá)，不受稻瘟病侵染誘導(dǎo)。Pi9的克隆為闡明廣譜抗病性的分子基礎(chǔ)以及水稻抗稻瘟病基因簇的進(jìn)化機(jī)制開辟了新的起點。
結(jié)果：通過對76kb的基因組片段進(jìn)行序列分析從Pi9位點鑒定出了一個NBS–LRR基因簇；序列比較和系統(tǒng)發(fā)生分析表明這6個Pi9 NBS–LRR基因?qū)儆?類旁系同源基因；對Pi9缺失突變體以及大片段DNA插入的分析將Pi9定位在含有Nbs2-Pi9和Nbs3-Pi9的基因組區(qū)域中；轉(zhuǎn)化單個候選基因確定Nbs2-Pi9為Pi9基因，同時轉(zhuǎn)化有Pi9的株系與Pi9的供體親本具有相同的廣譜抗病性；Pi9編碼一個NBS–LRR蛋白，長1032個氨基酸；Pi9在抗病植株中組成性表達(dá)。
討論：從R基因簇中圖位克隆Pi9基因；Pi9基因和蛋白的結(jié)構(gòu)特征；Pi9位點的基因組結(jié)構(gòu)；Pi9對稻瘟病的光譜抗病性的分子機(jī)制。