一个广谱持久抗稻瘟病基因pi-DY的克隆和功能分析

Rice blast is one of the three major rice diseases in China. The breeding of resistant varieties is an effective approach to prevent rice blast. However, R gene mediated blast resistance (vertical resistance) is easy to be overcame by the rice blast fungus. Discovery of slow-blasting resistance genes and study of the molecular mechanism are significance in rice blast resistance breeding. DaYu 23 is a broad-spectrum and durable slow-blasting resistant variety. It showed stable slow-blasting resistance in Guangxi blast field for many years, and showed stable slow-blasting resistance to the 185 single isolated diversity rice blast strains in greenhouse inoculation. The previous genetic analysis indicated that durable slow-blasting resistance is controlled by a recessive gene named pi-DY. Using a BC2F2:3 mapping population (from DaYu23×Nipponbare), we have localized the pi-DY locus to a 34kb interval in rice chromosome3. Knockout of one candidate gene(encoding an oxidoreductase) from the 34 kb region in Nipponbare significantly enhance its resistance to rice blast disease. In this project, function and mechanism of the gene will be revealed by using function complement experiment, spatio-temporal expression and subcellular localization pattern, etc. Knocking out pi-DY in hybrid rice parent 82B and R610 to explore the application value of slow-blasting resistance in hybrid rice breeding by gene editing. A better understanding of the function and the mechanism of pi-DY mediated rice blast resistance from this project will provide new insights for us to design durable rice blast resistance varieties in breeding.

稻瘟病是我国水稻三大病害之一。培育抗性品种是防治稻瘟病的有效途径，但垂直抗性品种的抗性易丧失。慢瘟抗性基因挖掘与分子机理研究，对于选育持久广谱抗稻瘟病的水稻品种有重要意义。大玉23是一个广谱持久的慢瘟抗性品种，多年多地病圃鉴定以及185个分离菌株的温室鉴定均表现稳定的抗性。前期遗传分析表明其持久抗性由隐性基因控制，命名为pi-DY，进一步构建大玉23（抗）与日本晴（感）的BC2F2：3家系群体，将pi-DY定位在3号染色体上34kb区域，其中一个编码氧化还原酶的候选基因03g13390，敲除等位基因的日本晴抗性显著增强。本项目计划通过基因功能互补验证，基因时空表达特性，及亚细胞定位模式等试验进一步明确其功能和机理。并敲除杂交稻亲本82B和R610的pi-DY基因，探索该基因通过基因编辑产生的慢瘟抗性在杂交稻育种中的应用价值。本研究预期将将为培育水稻广谱持久稻瘟病育种提供新理论和新基因。

本项目挖掘了一个广谱抗稻瘟病基因，4号染色体上LABR12区域存在一个核苷酸结合位点富含亮氨酸的重复序列NLR基因簇。这个基因簇中有一个基因在多个部分抗性水稻栽培种中高度保守，并且在稻瘟病病菌侵染的早期阶段，该基因的表达显著上调。鉴定了一个新的广谱抗性R基因，命名为PiPR1。.我们鉴定了两个稻瘟病易感基因，RNG1(Os02g39360)和RNG3(Os08g29170)，这两个基因的表达变异是由于它们3'-非翻译区(3'-UTR)中的多态性。这些多态性可以作为分子标记来鉴定对稻瘟病具有抗性的水稻种质。CRISPR-Cas9基因编辑技术介导的3'-UTR修改或敲除RNG1或RNG3中的任何一个基因，都能提高水稻对稻瘟病的抗性，而不影响五个关键的农艺性状。.根据Pi1与Piks、Pikh、Pikm、Pikp、Pik、Pi7基因序列之间的SNP开发了荧光分子标记MM-Pi1,并验证了它的特异性。分子标记辅助，聚合PiPR1、Pi1和Pi2，先后选育了三基因聚合的三系不育系宏泰A，软丰A和秋香A，并审定了聚合PiPR1、Pi1和Pi2杂交稻组合宏泰优621和软丰优610，秋香优302将于2023年审定。