水稻OsRDR5基因功能及作用机制初步解析

doi:10.16819/j.1001-7216.2025.250208

摘要/Abstract

摘要：

【目的】干旱是影响水稻生产的重要因素，避旱性是作物抗旱最重要的机制。研究水稻避旱性候选基因OsRDR5的作用机制，可以为水稻避旱性的改良提供理论依据。【方法】利用NCBI数据库对OsRDR5核酸序列、蛋白结构域和系统进化关系进行分析；采用qRT-PCR进行组织表达模式分析；通过CRISPR/Cas9系统构建OsRDR5基因敲除突变体；采用“篮子法”评价OsRDR5对水稻深根比的影响。【结果】OsRDR5具有一个Coa3_cc结构域，属于细胞色素c氧化酶组装因子3基因家族，定位于内质网，在水稻根、茎、叶等主要组织中均有表达。深根比表型鉴定发现，野生型深根比为53.7%，突变体株系深根比分别为63.3%和64.9%，显著高于野生型。根系转录组分析共鉴定到1434个差异表达基因。GO富集分析表明，这些基因显著富集到响应缺水、脱落酸、活性氧等与干旱胁迫相关的通路。在缺水通路中，多个与干旱胁迫相关的NAC基因(OsNAC016、OsNAC45等)上调表达。【结论】OsRDR5基因敲除影响了水稻深根比，在水稻避旱性中发挥重要作用。

关键词: 水稻, OsRDR5, 基因功能, 避旱性

Abstract:

【Objective】 Drought is a major constraint on rice production, and drought avoidance is the most important mechanism for drought resistance of crops. Understanding the mechanism of OsRDR5, a candidate gene of drought tolerance in rice, can provide a theoretical basis for the improvement of drought tolerance in rice.【Method】 The nucleotide sequence, protein domain and phylogenetic relationship of RDR5 were analyzed using the NCBI database. The tissue expression pattern was analyzed by qRT-PCR. An OsRDR5 gene knockout mutant was constructed by the CRISPR/Cas9 system. The effect of OsRDR5 on rice deep root ratio was evaluated by the "basket method".【Result】OsRDR5 has a Coa3_cc domain, belonging to the cytochrome c oxidase assembly factor 3 gene family. OsRDR5 is located in the endoplasmic reticulum and is expressed in roots, stems and leaves of rice. Phenotypic identification of deep root ratio showed that the wild type had a deep root ratio of 53.7%, while the mutant lines had a deep root ratio of 63.3% and 64.9%, respectively. The deep root ratio of the mutant was significantly increased. A total of 1434 differentially expressed genes were identified by root transcriptomic analysis. GO enrichment analysis revealed significant enrichment of these genes in pathways related to drought stress, including response to water deprivation, abscisic acid and reactive oxygen species, among which several NAC genes(OsNAC016, OsNAC45, etc.) related to drought stress were up-regulated in the water deprivation pathway.【Conclusion】OsRDR5 knockout affects the deep root ratio of rice and plays an important role in drought avoidance of rice.

Key words: rice, OsRDR5, gene function, drought avoidance

侯桂花, 周立国, 雷建国, 陈虹, 聂元元. 水稻OsRDR5基因功能及作用机制初步解析[J]. 中国水稻科学, 2025, 39(6): 779-788.

HOU Guihua, ZHOU Liguo, LEI Jianguo, CHEN Hong, NIE Yuanyuan. Preliminary Analysis of Function and Mechanism of OsRDR5 Gene in Rice[J]. Chinese Journal OF Rice Science, 2025, 39(6): 779-788.

图/表 12

表1 本研究使用的引物

Table 1. Primers used in this study

用途 Purpose	引物名称 Primer name	引物序列(5′-3′) Primer sequence (5′-3′)
基因克隆 Gene cloning	OsRDR5-1F	ATGGACGACGACGACCATG
基因克隆 Gene cloning	OsRDR5-1R	TTAGGATCCCGCGGTGGA
转基因鉴定 Identification of transgenes	OsRDR5-2F	TATGTTTGTCCGCCCCTCTG
转基因鉴定 Identification of transgenes	OsRDR5-2R	GAGAGAGTGGGGTGGAGGAA
组织表达模式分析 Analysis of tissue expression patterns	OsRDR5-qRT-3F	CCCTCTATACTGAACCGATGAC
组织表达模式分析 Analysis of tissue expression patterns	OsRDR5-qRT-3R	TAAAACTGCAATCCAACTCTGC
亚细胞定位 Subcellular localization	OsRDR5-4F	ATGGACGACGACGACCATGACC
亚细胞定位 Subcellular localization	OsRDR5-4R	AGATCCTCCTCCAGATCCTCCTC
内参基因Actin1 Reference gene Actin1	Actin1-F	CATCTATGAAGGATATGCTCTC
内参基因Actin1 Reference gene Actin1	Actin1-R	CCGTTGTGGTGAATGAGT
LOC_Os01g01430 qRT-PCR	qRT-F	TACTGCTGGGCGTGAAGAAG
LOC_Os01g01430 qRT-PCR	qRT-R	GCGTCTGCGGCTCAAATATG
LOC_Os03g04070 qRT-PCR	qRT-F	AGTGGTACTTCTTCGTGCCG
LOC_Os03g04070 qRT-PCR	qRT-R	CGTGACGTCTGCTTCTTCCT
LOC_Os11g03370 qRT-PCR	qRT-F	TATGGAGGCAGCAGCAACAA
LOC_Os11g03370 qRT-PCR	qRT-R	AGCTACAGGAGGAGGTGGAG
LOC_Os08g37370 qRT-PCR	qRT-F	GAAGTGGACCCAGGAGAACG
LOC_Os08g37370 qRT-PCR	qRT-R	ATGAACCCCTTGTACAGCGC
LOC_Os11g05614 qRT-PCR	qRT-F	ATCTCCTCCGCCACAAACAG
LOC_Os11g05614 qRT-PCR	qRT-R	GCCATGATCCGACGATGCTA
LOC_Os01g64310 qRT-PCR	qRT-F	ATTCACCACGAGATGCTGGG
LOC_Os01g64310 qRT-PCR	qRT-R	GCCACCTTGATCCATCAGCT
LOC_Os02g26720 qRT-PCR	qRT-F	CCTCCAGGAGTTCGTCAACC
LOC_Os02g26720 qRT-PCR	qRT-R	TTAGACCGATACCCAGCCCA

图1 OsRDR5基因的克隆

Fig. 1. Cloning of OsRDR5 M, DL5000 DNA Marker.

图2 OsRDR5基因结构域分析粉红色方块代表单跨膜区；蓝色方块代表结构域。

Fig. 2. Analysis of the domain of OsRDR5 gene Pink squares indicate single transmembrane regions; Blue squares indicate domains.

图3 RDR5同源蛋白的氨基酸序列多重比对粳稻：Oryza_sativa_Japonica；大麦：Hordeum_vulgare；画眉草：Eragrostis_curvula；柳枝稷：Panicum_virgatum；黍： Panicum_miliaceum；乌拉尔图小麦：Triticum_urartu；白福尼奥米：Digitaria_exilis；芦苇：Phragmites_australis；穇子： Eleusine_coracana_subsp._coracana ；谷子： Setaria_italica；玉米：Zea_mays；扫帚黍：Dichanthelium_oligosanthes。

Fig. 3. Multiple alignment of amino acid sequences of RDR5 homologous proteins

图4 RDR5 蛋白系统进化树

Fig. 4. Phylogenetic tree of RDR5 protein

图5 OsRDR5 的组织表达模式 A：OsRDR5芯片数据表达量；OsRDR5基因信号强度：Signal intensity；营养生长期：Vegetative；生殖生长期：Reproductive；成熟期：Ripening；叶片：Leaf blade；叶鞘：Leaf sheath；根：Root；茎：Stem；胚：Embryo；胚乳：Endosperm；14DAF、42DAF分别代表发育14 d 和42 d的胚和胚乳；12:00表示组织取样时间；B：以Actin1作为内参基因，数值为含标准误差的平均值，3次生物学重复。

Fig. 5. Tissue-specific expression of OsRDR5 A, OsRDR5 chip data expression level; Signal intensity, OsRDR5 gene signal intensity; Vegetative, Vegetative growth period; Reproductive, Reproductive growth period; Ripening, Ripening period; 14DAF and 42DAF represent embryos and endosperms after 14 days and 42 days of development, respectively; 12:00 indicates tissue sampling time; B, Actin1 gene was used as an internal control. Values are means±SE, n = 3.

图6 OsRDR5的亚细胞定位分析从左到右依次为荧光通道、标记通道、可见光通道、叠加图像；eGFP：RDR5-eGFP绿色荧光蛋白；ER：内质网, ER marker：SPER-mKATE红色荧光蛋白；Bright field：可见光通道；Merged：叠加图像；比例尺=10 μm。

Fig. 6. Subcellular localization of OsRDR5 From left to right, the fluorescence channel, marker channel, bright field and overlay images are shown; eGFP, RDR5-eGFP green fluorescent protein; ER, Endoplasmic reticulum; ER marker, SPER-mKATE red fluorescent protein; Bright field, Photographs captured under bright lighting conditions; Merged, An image of the superposition of three channels: eGFP, ER marker and Bright field; Scale bar =10 μm.

图7 OsRDR5基因序列分析 A：OsRDR5基因结构及突变位点分析。OsRDR5基因序列中黑色线条代表内含子，黑色条块代表外显子，黑色线条框代表UTR；WT为野生型，rdr5-1和rdr5-2为两个突变体；显示的序列均为靶位点及PAM位点；突变体序列中红色横线表示碱基缺失；B：蛋白质序列比对分析。WT编码185个氨基酸，rdr5-1编码35个氨基酸，rdr5-2编码24个氨基酸，完全相同的氨基酸用蓝色阴影表示。

Fig. 7. Sequence analysis of OsRDR5 gene A, Analysis of OsRDR5 gene structure and mutation sites. In the OsRDR5 gene sequence, black lines indicate introns, black blocks indicate exons, and black boxes indicate UTRs; WT was the wild type, rdr5-1 and rdr5-2 were two mutants; All the sequences displayed were the target sites and PAM sites; Red horizontal lines in the mutant sequence indicate base deletions; B, Protein sequence alignment analysis. WT encodes 185 amino acids, rdr5-1 encodes 35 amino acids, and rdr5-2 encodes 24 amino acids. Identical amino acids are shaded in blue.

A：野生型和rdr5株系的深根比及根系分布。深根比=深根数/总根数，数据显示为平均值±标准差，所有株系均有12个植株；*,**分别表示独立样本t检验下0.05，0.01水平上显著相关。

图8 WT和rdr5突变体的深根比

Fig. 8. Ratios of deep rooting of WT and rdr5 mutants A, Deep root ratio and root distribution map of WT and rdr5 lines. The deep root ratio = Number of deep roots/total number of roots; Data are shown as mean ± SD, (n = 12); *,** indicate significance at levels of 0.05, 0.01 by independent t-test, respectively.

图9 WT和rdr5突变体根系转录组分析 A：差异基因设置条件：P ≤ 0.05 和 |log2FC| ≥ 1；红色代表上调基因，蓝色代表下调基因；B：差异表达基因的qRT-PCR验证分析；rdr5-1和rdr5-2株系的qRT-PCR和RNA-Seq数值均以WT为校验标准，其中转录组测序数据计算公式为rdr5-1（FC）=rdr5-1（FPKM）/WT（FPKM）, rdr5-2（FC）=rdr5-2（FPKM）/WT（FPKM）；C：GO富集分析；D：KEGG富集分析。横坐标代表相应条目中基因占该条目所有基因的比例，纵坐标代表不同的基因功能条目；圆圈大小代表富集在相应条目中的基因数目，圆圈越大，代表富集在该通路中的基因越多；颜色代表富集显著性，圆圈表示该基因功能既关联到上调基因，也关联到下调基因，上三角形表示仅关联到上调基因，下三角形表示仅关联到下调基因，颜色代表富集显著性。

Fig. 9. Transcriptome analysis of WT and rdr5 mutant roots A, Differentially expressed genes: P≤0.05 and |log2FC|≥1; Red represents up-regulated genes and blue represents down-regulated genes; B, qRT-PCR verification analysis of differentially expressed genes; The qRT-PCR and RNA-Seq values of the rdr5-1 and rdr5-2 lines were measured using WT as the calibration standard, where the transcriptome sequencing data were calculated using the formula: rdr5-1（FC）=rdr5-1（FPKM）/WT（FPKM）, rdr5-2（FC）=rdr5-2（FPKM）/WT（FPKM）; C, GO enrichment analysis; D, KEGG enrichment analysis. The abscissa represents the proportion of genes in the corresponding item in all genes of the item, and the ordinate represents different gene function items; The size of the circle represents the number of genes enriched in the corresponding entry, and the larger the circle, the more genes enriched in the pathway; The color represents enrichment significance, the circle indicates that the gene function is associated with both up-regulated and down-regulated genes, the upper triangle indicates that it is only associated with up-regulated genes, the lower triangle indicates that it is only associated with down-regulated genes, and the color represents enrichment significance.

表2 响应干旱胁迫的差异表达基因

Table 2. Differentially expressed genes in response to drought stress

基因ID Gene ID	基因符号 Gene symbol	表达量差异倍数 log₂FC	P值 P-value	功能 Function
LOC_Os01g01430	OsNAC016	1.20	0.031	负调控水稻抗旱性Negative regulation of rice drought resistance
LOC_Os03g04070	ONAC022	1.40	0.0002	正调控水稻抗旱性Positive regulation of rice drought resistance
LOC_Os11g03370	OsNAC45	1.10	9.26E-06	正调控水稻抗旱性Positive regulation of rice drought resistance
LOC_Os01g64310	ENAC1	3.32	1.87E-12	干旱诱导表达基因Drought-induced expression gene

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