目的叶色突变相关基因鉴定和克隆有助于研究光合作用,补充并完善叶绿体发育机理和色素合成代谢途径,为开展水稻的高光效育种提供理论依据。方法从粳稻品种Dongjin的组培后代中分离出一个白条纹突变体st13,成熟期测定野生型和st13的主要农艺性状,苗期测定色素含量并观察叶绿体的超微结构;将st13和Dongjin进行正反交,观察F₁植株表型,并对F₂表型分离进行卡方检验,对st13进行遗传分析;利用st13×南京11（籼稻品种）的F₂和F_2:3群体,对st13突变基因定位;采用qPCR分析叶绿体发育和叶绿素合成相关基因在st13与野生型相对表达量。结果与野生型Dongjin相比,该突变体的株高、单株有效穗数、穗长、结实率和千粒重等主要农艺性状显著下降。苗期的色素含量降低,分蘖期无差异。突变体的叶绿体中既有含丰富的类囊体膜结构的正常叶绿体,也存在无类囊体结构的叶绿体。遗传分析和基因定位结果表明,st13的突变表型受1对隐性核基因控制,突变基因位于第3条染色体长臂InDel（Insertion-Deletion）标记I3-21和I3-22之间。进一步在这两个标记之间设计了6对InDel标记,最终将基因定位在94 kb区间内,此区间共有8个候选基因。结论这8个候选基因中,有5个假定的蛋白,其他三个都是有功能注释的蛋白,而这三个蛋白在水稻中均未见报道,因此,st13突变是由一个新的叶色基因突变引起的;同时st13中叶绿体发育、叶绿素合成和光合系统相关基因的表达也发生了显著改变,推测ST13可能是调控叶绿体发育的关键基因。

【Objective】Rice leaf color mutation identification and cloning of related genes is helpful to research photosynthesis and further understand the mechanism of chloroplast development and pigment synthesis pathway, providing theoretical basis for the breeding of rice with high photosynthetic efficiency.【Method】A somaclonal mutant (designated as stripe leaf 13, st13) was isolated from tissue-cultured progenies of a japonica rice variety Dongjin. The main agronomic traits of wild type and st13 were determined at maturity, and the pigment contents and ultrastructure of chloroplast were observed at the seedling stage. Dongjin and st13 were reciprocally crossed to observe the phenotype of F₁ and genetic analysis was carried out. An F₂ population and F_2:3 population derived from the cross st13 × Nanjing 11 (an indica rice variety) were used for gene mapping of ST13. Quantitative RT-PCR was carried out to analyze the relative expression of genes associated with chloroplast development and chlorophyll biogenesis in the wild-type Dongjin and st13 mutant. 【Result】Compared to the wild type, the plant height, number of effective panicles, panicle length, seed-setting rate, and 1000-grain weight were all significantly reduced in the mutant. In agreement with leaf color, the pigment content of st13 mutant was reduced at the seedling stage but rose to almost the same level as the wild type at the tillering stage. With a transmission electron microscopy, defective chloroplasts with no thylakoid were observed in the st13 mutant. Genetic analysis indicated the mutant phenotype was controlled by a single recessive nuclear gene. ST13 was restricted to an interval between the InDel (insertion/deletion) markers I3-21 and I3-22 on chromosome 3. Six InDel markers were further developed and the ST13 gene was narrowed down to a 94-kb region, containing eight putative open reading frames. 【Conclusion】These eight candidate genes encode three putative proteins and five functional proteins respectively, while none of them have been reported in rice. Thus, ST13 was an unreported gene in rice. Relative to wild type, the expression levels of genes associated with chloroplast development, chlorophyll biosynthesis, and photosynthetic system were significantly altered in the mutant. So we hypothesized that ST13 might be the key gene for regulating chloroplast development.