水稻叶色基因OsClpP6的功能研究
收稿日期: 2024-02-01
修回日期: 2024-03-21
网络出版日期: 2025-03-19
基金资助
科技创新2030—“智能设计育种技术创新与应用”重大项目(2023ZD04076);湖南省科技创新计划岳麓山种业创新项目(2021NK1002);长沙市科技计划资助项目(kq2004053)
Functional Characterization of Rice Leaf Color Gene OsClpP6
Received date: 2024-02-01
Revised date: 2024-03-21
Online published: 2025-03-19
【目的】探究OsClpP6在叶绿体发育过程中的功能，为提高植物光合速率提供新的基因资源。【方法】对OsClpP6基因、蛋白进行生物信息学分析，通过实时定量PCR和亚细胞定位技术分析该基因的表达模式，利用CRISPR/Cas9技术对该基因进行定点编辑，通过透射电镜观察突变体叶肉细胞叶绿体结构，利用RNA-seq分析OsClpP6影响叶色途径。【结果】Clp基因家族在水稻叶绿体早期发育中起重要作用，且在植物进化过程中十分保守。OsClpP6是水稻Clps基因家族的重要成员之一。通过研究OsClpP6的时空表达模式发现OsClpP6主要在苗期地上部分及营养生长期的茎中表达，水稻原生质体亚细胞定位结果表明，OsClpP6定位在叶绿体。在华占水稻背景下构建了OsClpP6的CRISPR/Cas9敲除突变株系clpp6-6s-ko-1、clpp6-6s-ko-2，突变体较WT株高降低、叶色变浅、千粒重减少。对WT、clpp6-6s-ko-1和clpp6-6s-ko-2灌浆期剑叶叶片进行透射电镜观察叶肉细胞的叶绿体微观结构发现，突变体叶肉细胞叶绿体结构、类囊体片层结构和淀粉粒积累明显少于WT。通过对突变体和WT进行转录组分析发现差异基因主要富集在光合作用通路，多个叶绿体发育的关键蛋白表达出现显著变化。【结论】OsClpP6通过参与叶绿体发育影响水稻源库的平衡，进而影响千粒重。
龚蒙萌, 宋书锋, 邱牡丹, 董皓, 张龙辉, 李磊, 李斌, 谌伟军, 李懿星, 王天抗, 雷东阳, 李莉 . 水稻叶色基因OsClpP6的功能研究[J]. 中国水稻科学, 2025 , 39(2) : 197 -208 . DOI: 10.16819/j.1001-7216.2025.240201
【Objective】This study aimed to investigate the function of OsClpP6 in chloroplast development, providing new genetic resources for improving plant photosynthetic rate. 【Method】Bioinformatics analyses were conducted to characterize the OsClpP6 gene and protein. The expression pattern of OsClpP6 was assessed through real-time quantitative PCR and subcellular localization techniques. Targeted editing of the OsClpP6 gene was carried out using CRISPR/Cas9 technology to generate targeted mutations in the OsClpP6 gene. The chloroplast structure in leaf mesophyll cells of mutants was examined using a transmission electron microscopy. Additionally, RNA-seq analysis was conducted to elucidate the impact of OsClpP6 on leaf color-related pathways. 【Result】The Clp gene family plays a crucial role in the early development of chloroplast in rice and is highly conserved during plant evolution. Oryza sativa L. caseinolytic protease P6 (OsClpP6) is an important member of the rice Clp gene family. Spatial and temporal expression pattern analysis revealed that OsClpP6 is predominantly expressed in the aerial parts of seedlings and stems during the vegetative growth stage, with subcellular localization in chloroplasts confirmed through experiments in rice protoplasts. CRISPR/Cas9-mediated knockout mutants of OsClpP6, clpp6-6s-ko-1 and clpp6-6s-ko-2, were generated in the background of Huazhan. These mutants exhibited phenotypic characteristics including reduced plant height, lighter leaf color, and decreased thousand-grain weight compared to the wild type. Transmission electron microscopy observation of flag leaves during the grain-filling stage revealed significantly fewer chloroplasts, thylakoid membrane layers, and starch granules in mutant leaf mesophyll cells compared to the wild type. Transcriptome analysis showed that differentially expressed genes were mainly enriched in the photosynthetic pathway, with significant changes in the expression of key proteins involved in chloroplast development. 【Conclusion】OsClpP6 participates in chloroplast development, affecting the balance of the rice source-sink relationship and subsequently influencing thousand-grain weight.
Key words： rice; leaf color; OsClpP6; Clp complex
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