水稻斑马叶突变体zl7的鉴定与基因的精细定位
收稿日期: 2022-03-28
修回日期: 2022-05-09
网络出版日期: 2023-03-10
基金资助
国家自然科学基金资助项目(31971872);国家自然科学基金资助项目(32171987);水稻生物学国家重点实验室自主课题(2020ZZKT10205);中国水稻研究所所级重点研发项目(CNRRI-2020-01);浙江省重点研发计划资助项目(2021C02056)
Identification and Gene Mapping of a Zebra Leaf Mutant zl7 in Rice
Received date: 2022-03-28
Revised date: 2022-05-09
Online published: 2023-03-10
【目的】斑马叶突变体作为水稻叶色突变体的重要种质资源，是研究植物光合作用机制和高光效育种的理想材料，对于解析光合作用机理和提高水稻产量具有重要意义。【方法】用甲基磺酸乙酯（EMS）诱变粳稻品种春江06建立突变体库，从突变体库中筛选到1份苗期为斑马叶的突变体，该突变体被命名为zl7 (zebra leaf 7)。在常规大田种植条件下分别比较突变体与野生型在苗期、抽穗期和成熟期叶色表型和产量性状差异，通过透射电镜实验分析叶片叶绿体发育情况，利用图位克隆方法克隆候选基因，利用荧光定量PCR 方法分析参与叶绿素合成和叶绿体发育相关基因的表达水平。【结果】从苗期开始，突变体zl7表现出典型的斑马叶，叶绿素含量降低，直到抽穗期，斑马叶表型消失，叶片逐渐复绿，叶绿素含量无明显差异。光合速率测定和电镜观察结果显示，突变体zl7的光合速率、气孔导度下降，叶绿体发育异常。与野生型相比，突变体的株高、分蘖、穗长、一次枝梗、二次枝梗和每穗粒数均显著降低，而粒长、粒宽和千粒重均略有增加。荧光定量PCR结果表明突变体中参与叶绿素降解相关基因的表达量显著升高，而参与叶绿素合成和叶绿体发育相关基因的表达量显著降低。遗传分析表明，该突变体受一对隐性核基因调控。通过图位克隆将该基因定位在第7染色体，测序发现突变体的目标基因ZL7编码区发生单碱基替换，导致一个氨基酸由丝氨酸变为天冬酰胺。【结论】ZL7 (Zebra Leaf 7)突变导致叶绿体发育异常，水稻叶片出现斑马叶表型，该基因在叶绿素合成及叶绿体发育中起重要作用。
廉院训, 韦子芸, 张强, 李清, 任德勇, 胡江, 朱丽, 高振宇, 张光恒, 郭龙彪, 曾大力, 钱前, 沈兰 . 水稻斑马叶突变体zl7的鉴定与基因的精细定位[J]. 中国水稻科学, 2023 , 37(2) : 113 -124 . DOI: 10.16819/j.1001-7216.2023.220314
【Objective】 Zebra leaf mutants, as important germplasm resources of rice leaf color mutants, are ideal materials for studying the mechanism of plant photosynthesis and high photosynthetic efficiency breeding, and play an important role in analyzing the mechanism of photosynthesis and improving rice yield. 【Method】 A zebra leaf mutant, designated as zebra leaf 7(zl7), was isolated from an ethyl methanesulfonate (EMS)-mutagenized population of Chunjiang 06 (CJ06). Under conventional paddy field management, the differences in leaf color phenotype and yield characters between the mutant and its wild type at seedling stage, heading stage and maturity stage were compared respectively. The development of leaf chloroplast were analyzed with a transmission electron microscope. Candidate genes were cloned by map-based cloning method, and the expression levels of genes involved in chlorophyll synthesis and chloroplast development were analyzed by fluorescence quantitative PCR. 【Result】From seedling stage, the mutant zl7 showed typical zebra leaves with decreased chlorophyll contents. During the heading stage, the zebra leaf phenotype disappeared and the leaves gradually turned green, and there was no significant difference in chlorophyll contents. The measurement of photosynthetic rate and the observation with an electron microscope showed that the photosynthetic rate, stomatal conductance and chloroplast development of the mutant zl7 were abnormal. Compared with the wild type, the plant height, the number of tillers, panicle length, primary rachis branch number, secondary rachis branch number and grains per panicle of the mutant decreased significantly, while the grain length, grain width and 1000-grain weight increased slightly. The results of fluorescence quantitative PCR showed that the expression levels of genes involved in chlorophyll degradation increased significantly, while the expression levels of genes involved in chlorophyll synthesis and chloroplast development decreased significantly. Genetic analysis showed that the mutant was regulated by a pair of recessive nuclear genes. The gene was mapped on chromosome 7 by map-based cloning, and the sequencing results showed that the coding region of ZL7 in zl7 has a single-base substitution mutation, causing an amino acid change from serine to asparagine. 【Conclusion】ZL7 mutation leads to abnormal chloroplast development, consequently resulting in zebra leaf phenotype, which plays an important role in rice chlorophyll synthesis and chloroplast development.
Key words： rice; chloroplast; genetic analysis; map cloning
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