基于WGCNA的稻谷储藏期间差异基因挖掘与品质调控网络构建

doi:10.3864/j.issn.0578-1752.2025.14.013

摘要/Abstract

摘要：

【背景】稻谷在长期储藏过程中经历复杂的生理和生化变化，这些变化不仅影响其储藏稳定性，还直接决定其最终的品质。储藏过程中，脂肪氧化、淀粉降解、蛋白质变化、膜稳态失衡及氧化胁迫等因素共同导致稻谷品质劣变。然而，稻谷储藏期间的分子机制尚不明确。【目的】本研究旨在通过WGCNA分析稻谷储藏期间的差异基因（DEGs），构建共表达网络，识别与稻谷储藏过程中品质变化密切相关的核心基因，并探讨其潜在调控机制，揭示稻谷储藏稳定性的关键分子基础。【方法】选取粳稻品种（南粳46号），分别在0、3、6、9和12个月的储藏时间点采样，并对样本进行转录组测序以获取基因表达数据。利用WGCNA分析筛选储藏期间差异较大的基因，构建加权基因共表达网络，并识别与储藏时间显著相关的特异性模块。基于网络连通性分析筛选核心基因，并结合富集分析、生理生化指标测定，以探索核心基因的功能。【结果】从基因表达谱中筛选出9 050个DEGs，其中8 654个基因在储藏不同阶段差异表达，396个为各储藏阶段共有的DEGs。WGCNA识别到17个基因共表达模块，进一步筛选出4个与稻谷储藏过程密切相关的特异性模块。网络连通性分析进一步挖掘出具有调控潜力的核心基因，包括参与脂质代谢的OsOLE4、OsCDAP3，维护细胞稳态的OsLEA32、OsAGP24、OsRHD3，乙烯信号通路调控基因OsERF064，以及控制表观遗传的OsEMF2a以及缺乏功能注释的5个候选基因。【结论】本研究基于转录组学和WGCNA技术，系统解析了稻谷储藏过程中的分子调控网络，表明稻谷通过多层次基因调控机制适应储藏环境。特定模块中的核心基因在抗氧化、营养代谢、膜稳态和细胞功能维持等方面发挥关键作用。

关键词: 稻谷, 储藏品质, 转录组测序, 加权基因共表达网络分析, 核心基因

Abstract:

【Background】Rice grain undergoes various physiological and biochemical changes during long-term storage, impacting both stability and quality. Those factors contribute to rice grain quality deterioration during its storage stage, such as lipid oxidation, starch degradation, protein modifications, membrane homeostasis imbalance, and oxidative stress collectively. However, the molecular mechanisms underlying these changes remain elusive.【Objective】This study aimed to analyze differentially expressed genes (DEGs) in stored rice grian, construct a co-expression network, identify core genes using WGCNA and explore regulatory mechanisms associated with rice storage stability. 【Method】Transcriptomic analysis was conducted on Japonica rice (Nanjing 46) grain stored for different durations (0, 3, 6, 9 and 12 months) to obtain gene expression profiles. WGCNA was employed to identify highly variable genes during storage, construct a weighted gene co-expression network, and identify storage-time-specific modules. Core genes screening was based on network connectivity, followed by functional enrichment analysis and physiological and biochemical assays to explore their potential roles in storage quality regulation.【Result】A total of 9 050 DEGs were identified, with 8 654 showing variations across storage stages, and 396 were expressed consistently across all time points. WGCNA identified 17 gene co-expression modules, of which four showed strong associations with storage duration. Connectivity analysis further highlighted key genes with regulatory potential：OsOLE4 and OsCDAP3, involved in lipid metabolism; OsLEA32, OsAGP24 and OsRHD3 associated with maintaining cellular stability; OsERF064 linked to the ethylene signaling pathway and OsEMF2a, an epigenetic regulator. Additionally, five candidate genes lacking functional annotation were identified for further study.【Conclusion】This study systematically analyzed the molecular regulatory network of rice grain storage using transcriptomics and WGCNA, revealing that rice grain adapts to storage environments through multi-level gene regulatory mechanisms. Core genes within specific modules played pivotal roles in antioxidant activity, nutrient metabolism, membrane stability, and cellular function maintenance. These findings provided a biological basis for delaying rice quality deterioration and offered potential genetic resources for improving rice grain storage stability.

Key words: rice grain (Oryza sativa), quality preservation, transcriptome sequencing, Weighted Gene Co-Expression Network Analysis (WGCNA), core genes

董雪, 陈梦秋, 邵晋, 吴学友, 唐培安. 基于WGCNA的稻谷储藏期间差异基因挖掘与品质调控网络构建[J]. 中国农业科学, 2025, 58(14): 2885-2903.

DONG Xue, CHEN MengQiu, SHAO Jin, WU XueYou, TANG PeiAn. Construction of a Differential Gene Expression and Quality Regulation Network in Stored Rice Grain Using WGCNA[J]. Scientia Agricultura Sinica, 2025, 58(14): 2885-2903.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2025.14.013

https://www.chinaagrisci.com/CN/Y2025/V58/I14/2885

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特异性模块GO功能富集"

模块 Module		基因数目 Gene number	GO编号 GO ID	类型 Term type	描述 Description	富集因子 Rich factor	P校正值 P-adjust
Brown		5	GO: 0008494	MF	翻译激活剂活性Translation activator activity	0.556	1.10E-04
		5	GO: 0097177	MF	线粒体核糖体结合Mitochondrial ribosome binding	0.417	5.59E-04
		5	GO: 0062125	BP	线粒体基因表达调控 Tegulation of mitochondrial gene expression	0.556	7.26E-04
		5	GO: 0070131	BP	线粒体翻译的正向调控 Positive regulation of mitochondrial translation	0.556	7.26E-04
		5	GO: 0070129	BP	线粒体翻译的调控Tegulation of mitochondrial translation	0.556	7.26E-04
		16	GO:0031974	CC	膜腔Membrane-enclosed lumen	0.083	2.98E-03
Blue		39	GO: 0045735	MF	营养库活动Nutrient reservoir activity	0.351	7.52E-28
		21	GO: 0033095	CC	糊粉粒Aleurone grain	0.600	1.09E-20
		14	GO: 0019865	MF	免疫球蛋白结合Immunoglobulin binding	0.483	1.88E-11
		14	GO: 0019863	MF	IgE 结合IgE binding	0.483	1.88E-11
		29	GO: 0005773	CC	液胞Vacuole	0.092	2.84E-05
		30	GO: 0044042	BP	葡聚糖代谢过程Glucan metabolic process	0.091	6.51E-05
Black		3	GO: 0004865	MF	蛋白丝氨酸/苏氨酸磷酸酶抑制剂活性 Protein serine/threonine phosphatase inhibitor activity	1.000	2.66E-04
		12	GO: 0006869	BP	脂质运输Lipid transport	0.067	1.23E-03
		8	GO: 0009873	BP	乙烯激活信号通路Ethylene-activated signaling pathway	0.094	3.57E-03
		7	GO: 0043621	MF	蛋白质自我结合Protein self-association	0.096	3.79E-03
		4	GO: 0012511	CC	单层环绕脂质贮存体 Monolayer-surrounded lipid storage body	0.400	4.16E-03
		4	GO: 0019915	BP	脂质储存Lipid storage	0.286	6.00E-03
Turquoise		1281	GO: 0005515	MF	蛋白结合Protein binding	0.239	3.23E-53
		841	GO: 0090304	BP	核酸代谢过程Nucleic acid metabolic process	0.260	1.12E-46
		2373	GO: 0043226	CC	细胞器Organelle	0.202	6.33E-43
		2366	GO: 0043229	CC	细胞内细胞器Intracellular organelle	0.202	7.62E-43
		2214	GO: 0043227	CC	膜结合细胞器Membrane-bounded organelle	0.204	7.62E-43
	2207	GO: 0043231	CC	细胞膜内细胞器Intracellular membrane-bounded organelle	0.204	1.55E-42

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基因 Genes	基因ID Gene ID	引物序列 Primer sequences（5′-3′)	产物大小 Amplicon size (bp)
EF-la	Os03g0177400	F: AGACCACCAAGTACTACTGCAC	540
EF-la	Os03g0177400	R: CCACCAATCTTGTACACATCC	540
Actin	Os04g0177600	F: AGACTACATACAACTCCATCAT	80
Actin	Os04g0177600	R: CACCACTGAGAACGATGT	80
OsEMF2a	Os04g0162100	F: ACTGCGTATTGTAAGGCTAA	268
OsEMF2a	Os04g0162100	R: CCGATGGCTATTATCAACTTC	268
-	Os12g0467200	F: ATGATGGTGAGGACGATTG	144
-	Os12g0467200	R: AGAAGATGTTGGAAGAAGCA	144
LGD1	Os09g0502100	F: GATGCCTCAGTGAACAATAG	238
LGD1	Os09g0502100	R: TAGTGCCTTCGGTCTCTT	238
-	Os02g0618250	F: GAATCTCCTCTCGGTGTTC	134
-	Os02g0618250	R: TACTTTGGTGCTGGCTTT	134
-	Os01g0212700	F: ACGGTGAGAAGCCAAGAA	144
-	Os01g0212700	R: GGTAGACGAGGAAGGAGAG	144
-	Os01g0342800	F: GAGAACTTCGTCAACTACC	105
-	Os01g0342800	R: GATCGAGTACTTGGAGAGG	105
OsCDAP3	Os07g0162900	F: CGTCTCGTCTCGTCTCAT	135
OsCDAP3	Os07g0162900	R: GGCGATGGATGGATGGAT
-	Os06g0563300	F: ATGAGGCGTCAGATTGTAA	178
-	Os06g0563300	R: CAGCACATGCGATAGAATT	178
OsLTP1.4	Os05g0477900	F: TGTTGGTTCTGGACTTCTG	170
OsLTP1.4	Os05g0477900	R: GATCGGACAAGCACACTT	170
OsC3H27	Os04g0394300	F: CCACAGCCATCTTCTTCC	152
OsC3H27	Os04g0394300	R: TTGGTGTCAGGAGGTAATG	152
OsS40-6	Os04g0413900	F: CCAGATGGTAAGGCAACAA	114
OsS40-6	Os04g0413900	R: CTCACAGTAATATCACACTCAC	114
OsLEA32	Os03g0747500	F: GACGAGGACAGGATCAAG	115
OsLEA32	Os03g0747500	R: ACACCATCCAAGGTTAGC	115

分类 Category	类型 Term type	GO ID GO编号	描述 Description	基因ID Gene ID	基因名 Gene name	功能注释 Gene description
上调 Up-regulate	MF	GO: 0003700	DNA 结合转录因子的活性 DNA-binding transcription factor activity	Os03g0183300	OsERF064	致病相关转录因子和含ERF结构域的蛋白质 Pathogenesis-related transcriptional factor and ERF domain containing protein
				Os01g0313300	OsERF068	类似EREBP-3蛋白（片段） Similar to EREBP-3 protein (Fragment)
				Os01g0657400	OsERF054	类似乙烯反应转录因子5（乙烯反应元件结合因子5） Similar to Ethylene-responsive transcription factor 5 (Ethylene-responsive element binding factor 5)
				Os05g0497300	OsERF074	类似乙烯反应因子2 Similar to Ethylene response factor 2
下调 Down-regulate	MF	GO: 0140657	ATP 依赖性活动 ATP-dependent activity	Os01g0533800	OsGSL4	保守假定性蛋白质 Conserved hypothetical protein
				Os06g0113150	OsGSL1	与 GSL8（GLUCAN SYNTHASE-LIKE 8）相似；1,3-β-葡聚糖合成酶/转移酶，转移糖基 Similar to GSL8 (GLUCAN SYNTHASE-LIKE 8); 1,3-beta-glucan synthase/ transferase, transferring glycosyl groups
				Os02g0832400	OsGSL7	与Callose synthase 1催化亚基相似 Similar to Callose synthase 1 catalytic subunit
				Os06g0728902	OsGSL6	类似预测蛋白质 Similar to predicted protein
	CC	GO: 0098797	质膜蛋白复合体 Plasma membrane protein complex	Os06g0158900	OsABCC15	类似耐多药相关蛋白3 Similar to Multidrug-resistance associated protein 3
				Os06g0184700	OsABCC12	保守假定性蛋白质 Hypothetical conserved gene
				Os03g0859500	OsABCG7	含ABC转运体类结构域的蛋白质 ABC transporter-like domain containing protein

模块 Module	基因数 Number	通路编号 Pathway ID	描述 Description	富集因子 Rich factor	P值 P value	P校正值 P-adjust
Brown	25	map03010	核糖体Ribosome	0.0568182	3.80E-06	0.0002773
Brown	5	map00790	叶酸的生物合成Folate biosynthesis	0.1250000	0.0012465	0.0454974
Black	12	map04141	内质网中的蛋白质加工 Protein processing in endoplasmic reticulum	0.0384615	5.04E-05	0.0028206
Blue	20	map00500	淀粉和蔗糖代谢 Starch and sucrose metabolism	0.0716846	6.45E-05	0.0056143
Turquoise	19	map00563	糖基磷脂酰肌醇（GPI）锚生物合成 Glycosylphosphatidylinositol (GPI)-anchor biosynthesis	0.5428571	1.64E-06	0.0001957
	28	map03440	同源重组Homologous recombination	0.3589744	0.0001526	0.0090771
	43	map03013	核细胞质运输Nucleocytoplasmic transport	0.2885906	0.0009115	0.0180778
	32	map00562	肌醇磷酸盐代谢 Inositol phosphate metabolism	0.3137255	0.0008698	0.0207011