The in silico map-based cloning of Pi36, a rice coiled-coil-nucleotide-binding site-leucine-rich repeat gene that confers race-specific resistance to the blast fungus
The in silico map-based cloning of Pi36, a rice coiled-coil-nucleotide-binding site-leucine-rich repeat gene that confers race-specific resistance to the blast fungus
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DOI：
10.1534/genetics.107.075465
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发表时间：
2007-08-01
期刊：
影响因子：
3.3
通讯作者：
Pan, Qinghua
中科院分区：
文献类型：
--
作者：
Liu, Xinqiong;Lin, Fei;Pan, Qinghua
籼稻品种Kasalath携带Pi 36基因，该基因决定对中国稻瘟病菌株的抗性，并已定位于8号染色体上的17 kb间隔。参考粳稻品种日本晴的基因组序列用于该区间的抗性（R）基因含量的二氧化硅预测，并因此用于鉴定Pi 36的候选基因。三个这样的序列，都有一个核苷酸结合位点和富含亮氨酸的重复基序，存在。通过长距离PCR从多个品种的基因组DNA中扩增这三个候选基因，并将所得扩增子插入pCAMBIA 1300和/或pYLTAC 27载体中以确定与抗性表型相关的序列多态性并进行转基因互补测试。将含有每个候选基因的构建体转化到稻瘟病敏感品种Q1063中，这允许鉴定Pi 36 -3为功能基因，另外两个候选基因是可能的假基因。P06编码的蛋白质由1056个氨基酸组成，在与抗性表型相关的残基590处具有单一取代事件（Asp至Ser）。Pi 36是水稻中的单拷贝基因，与大麦白粉病抗性基因Mla 1和Mla 6的关系比与稻瘟病抗性基因Pita、Pib、Pig和Piz-t的关系更密切。RT-PCR分析表明Pi 36在Kasalath中组成型表达。
The indica rice variety Kasalath carries Pi36, a gene that determines resistance to Chinese isolates of rice blast and that has been located to a 17-kb interval on chromosome 8. The genomic sequence of the reference japonica variety Nipponbare was used for an in silica prediction of the resistance (R) gene content of the interval and hence for the identification of candidate gene (s) for Pi36. Three such sequences, which all had both a nucleotide-binding site and a leucine-rich repeat motif, were present. The three candidate genes were amplified from the genomic DNA of a number of varieties by long-range PCR, and the resulting amplicons were inserted into pCAMBIA1300 and/or pYLTAC27 vectors to determine sequence polymorphisms correlated to the resistance phenotype and to perform transgenic complementation tests. Constructs containing each candidate gene were transformed into the blast-susceptible variety Q1063, which allowed the identification of Pi36-3 as the functional gene, with the other two candidates being probable pseudogenes. The P06-encoded protein is composed of 1056 amino acids, with a single substitution event (Asp to Ser) at residue 590 associated with the resistant phenotype. Pi36 is a single-copy gene in rice and is more closely related to the barley powdery mildew resistance genes Mla1 and Mla6 than to the rice blast Rgenes Pita, Pib, Pig, and Piz-t. An RT-PCR analysis showed that Pi36 is constitutively expressed in Kasalath.