【摘要】 稻瘟病是影响水稻(Oryza sativa)稳产的因素之一，类病斑突变体作为是作物抗病分子机制的理想材料，解析突变体的抗病基因是培育稳产抗病水稻的基础。本研究通过对粳稻品种’武运粳21’进行甲基磺酸乙酯(ethylmethylsulfone, EMS)诱变，获得1个稳定遗传的类病斑突变体spl52(spotted leaf 52)。成熟期突变体植株的有效穗数、每穗粒数、千粒重和结实率相比野生型显著降低。从分蘖初期开始突变体spl52叶尖部出现病斑。通过3,3’-二氨基联苯胺(3,3’-diaminobenzidine, DAB)染色、酶活测定和二氯荧光素二乙酸酯(2’,7’-dichlorodihydrofluorescein diacetate, H2DCFDA)检测表明，突变体spl52病斑叶片出现活性氧积累，通过TUNEL实验发现细胞程序性死亡激活。对野生型和突变体叶片进行透射电镜分析，结果显示，突变体叶绿体呈现较小的椭圆形，线粒体呈现空洞化。遗传分析表明，突变体的表型受1对隐性核基因控制，通过Mutmap分析，发现6个可能的候选区间，使用图位克隆技术将候选基因定位于6号染色体RM3.5～RM4.3。对候选基因进行分析，未发现已报道的类病斑及水稻抗性相关基因。对野生型和突变体植株叶片接种稻瘟病菌种(Magnaporthe oryzae)，结果表明，突变植株对稻瘟病菌更敏感。q RT-PCR结果显示，突变体中正向调控水稻抗性的基因OsPR1a、OsPR1b、JIOsPR10、OsWRKY6、OsRSR1表达量均显著低于野生型。结果表明，突变基因为尚未报道的新基因，参与了水稻稻瘟调控相关途径。更多还原

【Abstract】 Rice(Oryza sativa) blast is one of the factors affecting the stable yield of rice. The lesion mimic mutant is an ideal material for studying the molecular mechanism of crop disease resistance. The analysis of the disease resistance gene of the mutant is the basis for cultivating stable and disease-resistant rice. In this study, a stable genetic lesion mimic mutant spl52(spotted leaf 52) was obtained by ethylmethylsulfone(EMS)mutagenesis of japonica rice ’Wuyunjing 21’. The effective panicle number, grain number per panicle, 1000-grain weight and seed setting rate of the mutant plants at maturity stage were significantly lower than those of the wild type. From the early tillering stage, the leaf tip of mutant spl52 appeared disease spots. By 3, 3’-Diaminobenzidine(DAB) staining, enzyme activity assay and 2’, 7’-dichlorodihydrofluorescein diacetate(H2DCFDA) detection results showed that the mutant spl52 lesion leaves showed reactive oxygen species accumulation, and programmed cell death activation was found by TUNEL assay. Transmission electron microscopy analysis of wild-type and mutant leaves showed that the mutant chloroplasts were smaller oval,and the mitochondria were hollowed out. Genetic analysis showed that the phenotype of the mutant was controlled by a pair of recessive nuclear genes. Six possible candidate intervals were found by Mutmap analysis, and the candidate genes were located between RM3.5-RM4.3 on chromosome 6 by map-based cloning. The candidate genes were analyzed, and no reported lesion-like and rice resistance-related genes were found. The leaves of wild-type and mutant plants were inoculated with rice blast fungus, and the results showed that the mutant plants were more sensitive to rice blast fungus(Magnaporthe oryzae). The results of qRT-PCR showed that the expression levels of OsPR1a, OsPR1b, JIOsPR10, OsWRKY6 and OsRSR1 which positively regulated rice resistance in the mutant were significantly lower than those in the wild type. The results showed that the mutant gene was a new gene that had not been reported and was involved in the regulation of rice blast.更多还原