水稻类病斑突变体lmm7的鉴定与基因定位

Phenotypic Identification and Gene Mapping of a Lesion Mimic Mutant lmm7 in Rice

【目的】对水稻类病斑突变体的研究有助于解析其与植物生长和防御反应的关系。【方法】本研究在粳稻品系FI135胚培养过程中获得了1个类病斑突变体lmm7(lesion mimic mutant 7)。通过对其进行系统的表型鉴定、农艺性状考查、超微结构观察、生理学特性分析,阐明LMM7基因对植物生长的调控。通过病原菌抗性鉴定,明确lmm7对植物防御反应的影响。利用9311B与突变体lmm7杂交所得F2群体对该突变体进行了遗传分析和基因精细定位。【结果】该突变体苗期表型正常,分蘖初期,植株基部叶片从叶尖开始不断出现褐色斑点,并向整株扩散,且斑点数目随植株生长不断增加。与野生型相比,突变体的株高、穗长、有效穗数、每穗粒数、结实率及剑叶长宽都显著降低,但籽粒性状和抽穗期没有显著性差异。遮光处理表明,突变体lmm7的表型受到光照诱导,抽穗期突变体lmm7叶肉细胞严重失绿,光合色素含量显著降低。组织化学分析表明,突变体病斑处的H2O2含量显著升高。透射电镜观察结果表明,突变体lmm7叶肉细胞的叶绿体数目减少,叶绿体类囊体片层结构严重受损,细胞器肿胀解体,并出现大量嗜锇小体,同时病斑内部和周围区域积累了大量的ROS。抗性鉴定结果显示突变体lmm7稻瘟病抗性水平显著高于野生型。遗传分析表明lmm7的突变表型受单个隐性基因控制。利用图位克隆的方法,目的基因被定位于水稻第7染色体短臂两InDel标记7B35和7B43之间,区间范围约260 kb。测序结果表明该区间内候选基因LOC_Os07g0203700第2891位碱基T发生了单碱基缺失,导致后续移码突变及翻译提前终止。【结论】lmm7与spl5互为等位基因,其突变抑制了植株的生长,同时增强了对稻瘟病的抗性。

【Objective】The study of lesion mimic mutants(LMMs)is helpful to understand the interaction between growth and defense response in plants.【Method】We herein reported a novel lesion mimic mutant 7(lmm7)derived from a japonica rice variety FI135 by embryo culture.For deciphering the effect of lmm7 on plant growth,phenotype and agronomic traits were compared between the wild type(WT)and lmm7 plants.The ultrastructure of mesophyll cells and histochemical characterization was done in WT and lmm7 plants.Evaluation of WT and lmm7 was observed for resistance to pathogens was employed to explore the underlying effect of lmm7 on plant defense response.An F2 population derived from the cross between lmm7 mutant and 9311B was used for genetic analysis and gene mapping.【Result】The seedlings of lmm7 were normal at the seedling stage,but the basal leaves began to exhibit brown spots at the initial tillering stage,which then spread to the whole plant,and the number of mosaic spots increased along with the development of lmm7 plants.Compared with the wild type,agronomic traits including plant height,panicle length,effective panicle number,grain number per panicle,and seed setting rate were significantly decreased in the mutant.However,no significant difference in seed traits and heading date was observed between WT and lmm7 plants.The shading assay indicated that lesion formation in lmm7 was induced by light.The mesophyll cells of lmm7 mutant were seriously chlorotic,and the photosynthetic pigment contents of the mutant were significantly decreased at the heading stage.Histochemical analysis showed that a large amount of hydrogen peroxide(H2O2)accumulated at the lesion sites.The observation with a transmission electronic microscope(TEM)demonstrated that the number of chloroplasts in mesophyll cells of lmm7 plants was decreased,the lamellar structure of chloroplasts was seriously damaged,and osmiophilic granules were obviously increased in the mutant.Moreover,the levels of reactive oxygen species(ROS)were increased in the internal and surrounding regions of the lesions.The lmm7 plants displayed higher resistance to rice blast compared to the wild type.Genetic analysis revealed that in lmm7 the mutation was controlled by a single recessive gene.Using a map-based cloning strategy,lmm7 was narrowed down to a 260 kb region between InDel markers 7B35 and 7B43 on the short arm of chromosome 7 in rice.Sequencing analysis revealed that a deletion of T base at 2891 nucleotide position occurred in the DNA sequence of LOC_Os07g0203700,resulting in a frame-shift mutation and a premature stop codon.【Conclusion】The results reveal the target gene is allelic to spl5,and the mutation of lmm7 enhances resistance to rice blast,while it curbs the growth and development of lmm7 plants.