长雄野生稻抗稻瘟病新基因Pi57(t)的克隆

With many excellent characteristics for resistance to biotic and abiotic stresses, Oryza longistaminata is one of the important reservoirs of useful genes transferring to Asian cultivated rice (Oryza sativa). In previous studies, we have identified a novel gene, Pi57(t) conferring broad-spectrum resistance to rice blast from the introgression line E1454 of O. longistaminata. This gene was mapped on the short arm of chromosome 12 of rice, flanked by 2 SSR marker RM27921 and RM27943. However, for the structure, function as well as molecular mechanism of broad-spectrum resistance to rice blast of Pi57(t) gene remain unknown. In present studies, in orer to clarify the structure and function of Pi57(t) gene, map-based cloning strategy will be employed to clone this gene. The candidate resistance gene(s) (CRG) will be determined, by fine mapping of gene, prediction of CRGs in mapped region, expression anlysis of CRGs and comparing analysis of allelic sequences of CRGs in resistant cultivars and susceptible cultivars. Then, transformation vector carrying CRG will be constructed via amplification DNA sequences of CRGs from resistant donor E1454 based on PCR method. The CRGs will be transformed into the susceptible rice cultivars to determine the function for resistance to rice blast via agrobacterium-mediated transformation strategy. These results will lay the foundation for further researches on resistance mechanism of gene conferring broad-spectrum resistance to M. oryzae, gene evolution as well as interaction between resistance gene from rice and avirulence gene from M. oryzae, and will prvide important gene for transgenic rice breeding for resistance to rice blast.

长雄野生稻具有许多抗生物胁迫和非生物胁迫的优异特性，是向亚洲栽培稻转移有利基因的优良基因库之一。项目组前期从长雄野生稻渗入系E1454中鉴定到1个广谱抗稻瘟病的新基因Pi57(t)，该基因被初步定位到水稻第12染色体短臂上，位于2个SSR标记RM27921和RM27943之间。然而，对于该基因的结构、功能和广谱抗性分子机制尚不清楚。本项目以抗稻瘟病新基因Pi57(t)为研究对象，采用图位克隆的方法，通过基因的精细定位、候选基因预测及表达分析、候选基因在抗病和感病品种中的等位基因序列比较分析，确定候选抗病基因；采用PCR技术从抗病供体亲本中扩增候选基因，构建候选抗病基因的遗传转化载体；并通过农杆菌介导的遗传转化法开展抗病基因功能验证，完成该基因的克隆，阐明其结构与功能。研究结果将为开展广谱抗性机制、基因的演化以及基因互作机制的研究奠定基础，并为转基因抗病育种提供重要的基因源。

本项目利用长雄野生稻渗入系IL-E1454与亚洲栽培稻RD23以及丽江新团黑谷（LTH）杂交获得的39445个F2代群体，在开展基因型及表型分析的基础上，将来源于长雄野生稻的抗稻瘟病新基因Pi57(t)精细到水稻第12染色体短臂上51.7 kb的物理距离范围内，完成了该基因的精细定位；靶标区域的候选基因基因注释分析发现3个预测的编码基因；通过RT-PCR分析发现在IL-E1454中仅1个候选基因Os12g18729表达，该基因被确定为Pi57(t)的候选基因；转基因实验表明，经基因编辑及RNAi干涉的植株表现感病，而转入候选基因全长序列的感病品种表现抗病，从而证明候选基因Os12g18729就是Pi57(t)基因，该基因编码1个由855个氨基酸组成的蛋白，与目前已克隆的抗病基因具有不同的结构，该基因编码一个具有跨膜结构域和犰狳重复序列（Transmembrane-Armadillo repeat, TM-ARM）的蛋白，是一类全新的抗性蛋白；利用绿色荧光蛋白构建融合基因的亚细胞定位表明该基因编码的Pi57(t)蛋白被定位在细胞膜上表达；本项目成功克隆Pi57(t)基因，对进一步利用分子标记辅助选择和转基因技术开展稻瘟病抗性育种提供了重要的基因源，同时为进一步研究水稻与稻瘟病菌无毒基因的互作，阐明Pi57(t)基因的抗病机理，寻找新的稻瘟病控制途径提供了宝贵的研究素材。在项目执行期间发表SCI论文2篇，1人获得云南省中青年学术和技术带头人称号。