水稻雷帕霉素靶蛋白OsTOR调控根伸长的功能研究

doi:10.3864/j.issn.0578-1752.2026.03.001

摘要/Abstract

摘要：

【目的】克隆水稻雷帕霉素靶蛋白基因OsTOR，探究其在根伸长过程中的调控机制，解析其在水稻发育中的生物学功能，为改良水稻性状、提高产量提供分子依据。【方法】以粳稻品种Dongjin为材料，运用实时荧光定量PCR分析OsTOR的表达模式；利用RT-PCR技术克隆OsTOR，构建35S:OsTOR-GFP载体，通过农杆菌介导的水稻遗传转化，创制过表达植株OsTOR-OX，观察OsTOR的亚细胞定位；用TOR活性抑制剂AZD-8055处理水稻幼苗并观察表型；对抑制剂处理植株和OsTOR-OX植株根尖进行透明处理，激光共聚焦显微镜下观察分生区细胞形态；取萌发一致的未处理和处理后的7日龄水稻幼苗进行转录组测序并分析差异基因；利用实时荧光定量PCR对根尖分生组织中的细胞周期相关基因进行表达分析。【结果】OsTOR在进化上与大麦和小麦较近，包括HEAT重复区、FAT结构域、FRB结构域、PIKKc激酶域和FATc结构域，定位于细胞质和细胞膜，表达于萌发后7 d的水稻幼苗叶片、叶鞘、根、成熟植株的旗叶叶片、旗叶叶鞘、花序、成熟花药，以及即将授粉的雌蕊中。经抑制剂AZD-8055处理后，幼苗生长及根的伸长受到抑制，根尖分生区细胞增殖能力降低。过表达OsTOR则促进幼苗根的伸长。转录组测序结果表明，经AZD-8055处理后，有225个基因被上调，121个基因被下调。上调基因中，约28%参与细胞活动，18.6%参与对刺激响应，还有部分基因参与物质降解等过程；下调基因主要包括生物合成、蛋白质代谢以及细胞周期相关的基因。qRT-PCR结果表明，OsTOR活性降低导致细胞周期相关基因表达下降。【结论】OsTOR定位于细胞质和细胞膜，在水稻不同发育时期、不同组织中均有表达；OsTOR活性降低抑制水稻幼苗生长及根的伸长，过表达OsTOR则促进根的伸长，主要由于细胞周期相关基因的变化导致根尖分生区细胞增殖受到影响，进而影响根的伸长。

关键词: 水稻, 雷帕霉素靶蛋白, 根, 分生组织, 细胞周期

Abstract:

【Objective】To clone the target of rapamycin gene OsTOR in rice, and investigate its regulatory mechanism in root elongation, elucidate its biological function in rice development, and provide a molecular basis for improving rice traits and increasing its yield. 【Method】In this research, the japonica rice variety Dongjin was used as material for OsTOR expression pattern analysis via qRT- PCR. OsTOR was cloned by RT-PCR, and the overexpression vector 35S:OsTOR-GFP was constructed. The overexpression lines OsTOR-OX were generated by Agrobacterium-mediated rice genetic transformation to examine the subcellular localization of OsTOR. Rice seedlings were treated with TOR inhibitor AZD-8055, and the phenotype was observed. The root tips of inhibitor-treated and OsTOR-OX plants were cleared and the cell morphology in the meristematic zone was then visualized by CLSM. Transcriptome sequencing was performed on 7-day-after-germination (DAG) seedlings with or without AZD-8055 treatment. The expression of cell cycle related genes was further analyzed by qRT-PCR. 【Result】OsTOR was evolutionally closely related to barley and wheat TORs. It contains HEAT repeat domain, FAT, FRB, PIKKc kinase domain and FATc domain. OsTOR was localized in cytoplasm and cell membrane, and expressed in various tissues: leaves, leaf sheaths and roots of 7 DAG seedlings, as well as flag leaf blades, flag leaf sheaths, inflorescences, mature anthers, and unpollinated pistil of mature plant. AZD-8055 inhibited seedling growth and root elongation, accompanied by reduced cell proliferation in the root meristem. In contrast, OsTOR overexpression promoted root elongation. Transcriptome analysis revealed 225 genes were upregulated and 121 genes were downregulated upon AZD-8055 treatment. Among the upregulated genes, approximately 28% were associated with cell process, 18.6% with response to stimulus, and some others were involved in degradation. Downregulated genes were mainly involved in biosynthetic processes, protein metabolism, and cell cycle regulation. qRT-PCR results further confirmed that reduced OsTOR activity led to decreased expression of cell cycle-related genes. 【Conclusion】OsTOR was localized in cytoplasm and cell membrane, and was ubiquitously expressed in various tissues at different developmental stages of rice. Reduced OsTOR activity restrained seedling growth and root elongation, whereas OsTOR overexpression promoted root elongation. This effect was primarily attributed to altered expression of cell cycle-related genes, which subsequently affected cell proliferation in root meristem.

Key words: rice, target of rapamycin, root, meristem, cell cycle

朱姝, 国志鹏, 孙颖. 水稻雷帕霉素靶蛋白OsTOR调控根伸长的功能研究[J]. 中国农业科学, 2026, 59(3): 475-485.

ZHU Shu, GUO ZhiPeng, SUN Ying. Functional Analysis of Rice Target of Rapamycin OsTOR in Regulating Root Elongation[J]. Scientia Agricultura Sinica, 2026, 59(3): 475-485.

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基因 Gene	基因号 GeneID	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
OsTOR	LOC_Os05g14550	TTCTTGCTGAAAGGCCATGA	CCCGATTAGCAGCTTCTTCG
CDC6	LOC_Os01g63710	GAGATCCCGCTCAGATGCAGG	GTCTTCCCTGTACCAGGGCAC
MCM5	LOC_Os02g55410	CTCCGCAAGTTCAAGGAGTTC	CAGCGGTAGGTAATCAGCGG
ORC2	LOC_Os03g08640	GCAAGTCCATGTTGTCGCATC	GGCTTTGCAGAACAACGAGG
ORC6	LOC_Os07g43540	CCAAGTTCCAGGTGATATTCGAC	CCTGACACAGCCGAACTGG
UBQ	LOC_Os01g48280	CTAAGTGCACCAAACCCTGATG	TCTGCTTCATTGGCTTTCCA