水稻白化致死突变体wll2的基因克隆与功能研究

doi:10.3864/j.issn.0578-1752.2026.17.001

摘要/Abstract

摘要：

【目的】探究甲基赤藓糖醇磷酸途径（methylerythritol phosphate，MEP）关键基因对叶绿体发育、叶绿素合成和幼苗生长的调控机制，阐明水稻叶色突变体白化致死的分子机理，为水稻光合机制解析与高光效种质创新提供理论依据。【方法】以野生型武运粳8号和白化致死突变体wll2（white leaf and lethal 2）为研究对象，综合运用色素测定、透射电镜技术、MutMap+、CRISPR-Cas9基因编辑技术、qRT-PCR技术、烟草瞬时表达、转录组测序分析和Western blot等分子生物学手段克隆目的基因并解析其功能。【结果】wll2从种子萌发开始即出现明显的叶片白化表型，且持续稳定，直至幼苗生长至四叶期死亡。wll2幼苗生长受到严重抑制，其苗长和根长显著低于野生型。与野生型相比，wll2的叶绿素a和叶绿素b含量显著降低，其叶肉细胞中叶绿体数量显著减少，内部类囊体片层数量减少、排列紊乱。利用MutMap+和基因编辑技术，确定导致叶片白化表型的基因为MEP萜类合成途径中的1-脱氧-D-木酮糖-5-磷酸合成酶基因OsDXS1。qRT-PCR分析结果表明，OsDXS1在水稻不同组织中均有表达，其中，在叶片中的表达量最高，显著高于根、茎、叶鞘和幼穗。烟草瞬时表达试验显示，OsDXS1编码蛋白的荧光信号与叶绿体自发荧光完全重叠，证实其定位于叶绿体，与其参与MEP途径的功能相符。转录组测序共鉴定出7 197个差异表达基因（DEGs），其中，上调基因4 055个、下调基因3 142个。进一步GO与KEGG富集分析表明，下调DEGs主要富集于光合作用、质体功能和相关代谢通路，而上调DEGs聚焦于细胞周期、DNA代谢、核酸复制、物质代谢重塑和损伤修复等适应性响应通路。Western blot检测显示，光合系统Ⅱ核心蛋白PsbE和PsbO、光系统Ⅰ核心蛋白PsaA，以及光捕获复合体组分LHCa1的含量均显著降低。【结论】水稻白化致死突变体wll2的突变基因为MEP途径关键基因OsDXS1，该基因功能缺失会导致叶绿素合成受阻、叶绿体发育异常并引发白化致死。

关键词: 水稻, 白化致死, MutMap+, OsDXS1, 叶绿体, 转录组测序

Abstract:

【Objective】This study aims to investigate the regulatory mechanism of key gene in the methylerythritol phosphate (MEP) pathway on chloroplast development, chlorophyll biosynthesis, and seedling growth in rice. It seeks to elucidate the molecular mechanism underlying the albino lethal phenotype of the rice leaf color mutant, thereby providing a theoretical foundation for understanding photosynthetic mechanisms and developing germplasm with high photosynthetic efficiency.【Method】The wild-type rice variety Wuyunjing 8 and its derived albino lethal mutant wll2 (white leaf and lethal 2) were used as experimental materials. A combination of molecular biology techniques, including pigment content determination, transmission electron microscopy, MutMap+ analysis, CRISPR-Cas9 gene editing, quantitative real-time PCR (qRT-PCR), transient expression assay in tobacco leaves, transcriptome sequencing (RNA-seq), and Western blot analysis, was employed to clone the target gene and characterize its function.【Result】The wll2 mutant exhibited a distinct albino phenotype from germination, which persisted until the seedlings died at the four-leaf stage. Seedling growth was severely inhibited, with significantly reduced shoot and root lengths compared to the wild type. The mutant showed significantly lower chlorophyll a and chlorophyll b contents. Mesophyll cells of wll2 contained significantly fewer chloroplasts, with reduced and disorganized thylakoid membranes. Using MutMap+ analysis combined with gene editing, the gene responsible for the albino phenotype was identified as OsDXS1, which encodes 1-deoxy-D-xylulose-5-phosphate synthase, a key enzyme in the MEP terpenoid biosynthesis pathway. qRT-PCR analysis revealed that OsDXS1 is expressed in various rice tissues, with the highest transcript level in leaves, significantly exceeding that in roots, stems, sheaths, and young panicles. Transient expression in tobacco demonstrated that the OsDXS1 protein co-localized with chloroplast autofluorescence, confirming its chloroplast localization, consistent with its role in the MEP pathway. Transcriptome analysis identified 7 197 differentially expressed genes (DEGs) between wll2 and the wild type, comprising 4 055 up-regulated and 3 142 down-regulated genes. GO and KEGG enrichment analyses revealed that down-regulated DEGs were primarily enriched in photosynthesis, plastid function, and related metabolic pathways. Conversely, up-regulated DEGs were enriched in adaptive response pathways, including cell cycle, DNA metabolism, nucleic acid replication, metabolic reprogramming, and damage repair. Western blot analysis showed significantly reduced protein levels of the photosystem Ⅱ core proteins PsbE and PsbO, the photosystem I core protein PsaA, and the light-harvesting complex component LHCa1 in the mutant.【Conclusion】The gene responsible for the albino lethal phenotype in the rice wll2 mutant is OsDXS1, a key gene in the MEP pathway. Loss of function of OsDXS1 disrupts chlorophyll biosynthesis and impairs chloroplast development, ultimately leading to the albino lethal phenotype.

Key words: Oryza sativa, albino lethal, MutMap+, OsDXS1, chloroplast, RNA-seq

张晔, 王程程, 张龙. 水稻白化致死突变体wll2的基因克隆与功能研究[J]. 中国农业科学, 2026, 59(17): 3699-3711.

ZHANG Ye, WANG ChengCheng, ZHANG Long. Gene Cloning and Functional Characterization of the Albino Lethal Mutant wll2 in Rice[J]. Scientia Agricultura Sinica, 2026, 59(17): 3699-3711.

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