基于WGCNA鉴定大豆抗大豆花叶病毒NAC转录因子及其诱导表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0109

摘要/Abstract

摘要：

目的挖掘大豆抗大豆花叶病毒（soybean mosaic virus, SMV）病相关的核心转录因子（transcription factors, TFs），为深入解析大豆抗病分子机制和创制抗病品种提供理论依据。方法以大豆抗病材料X149和感病材料X97受大豆花叶病毒株系SC15诱导后的转录组数据为基础，利用PlantTFDB v 5.0数据库从转录组中预测全基因组转录因子基因；基于差异表达转录因子基因，通过加权基因共表达网络分析（weighted gene co-expression network analysis, WGCNA）获得抗病转录因子相关模块和抗病核心转录因子；采用String 12.0预测转录因子互作蛋白；利用RT-qPCR分析转录因子受激素（ETH、SA、MeJA和ABA）诱导表达情况。结果全基因组含有转录因子的基因有3 170个，其中1 727个属于差异表达基因；WGCNA分析表明1 727个基因被划分为6个转录因子基因共表达模块，其中brown模块和turquoise模块与X149抗性显著相关，2个模块中转录因子基因间连通度最高的核心转录因子为turquoise模块中NAC转录因子GmNAC030基因，turquoise模块中与GmNAC030具有相似表达的NAC转录因子基因有GmNAC043、GmNAC085、GmNAC092和GmNAC101；互作蛋白预测表明，GmNAC030、GmNAC043和GmNAC092互作蛋白均为转录因子，且仅有互作蛋白Glyma.02g131700（bZIP1）、Glyma.16g164800（AP2-EREBP）和Glyma.08G118200（WRKY48）位于模块中。RT-qPCR分析表明GmNAC030主要受MeJA诱导，另外4个NAC转录因子基因主要受MeJA和ETH诱导。结论 5个NAC转录因子基因GmNAC030、GmNAC043、GmNAC085、GmNAC092和GmNAC101的表达与大豆抗SMV相关且受外源激素MeJA和ETH的诱导。

关键词: 大豆, 大豆花叶病毒, 加权共表达网络分析, NAC转录因子, 表达分析

Abstract:

Objective To identify the hub genes of transcription factors associated with soybean resistance to soybean mosaic virus (SMV), which may provide a theoretical foundation for elucidating molecular mechanism and developing resistant germplasm. Method Using the transcriptome data of soybean disease-resistant material X149 and disease-susceptible material X97 induced by SMV SC15, PlantTFDB v5.0 database was used to predict the transcription factor genes of the whole genome. Weighted gene co-expression network analysis (WGCNA) was applied to identify the resistance-associated modules and hub transcription factors based on differentially expressed transcription factor genes. String 12.0 was for predicitng transcription factor interacting proteins. RT-qPCR was to analyze the expression patterns of transcription factor genes induced by hormones (ETH, SA, MeJA and ABA). Result The whole genome contained 3 170 genes encoding transcription factors, of which 1 727 were differentially expressed. WGCNA analysis revealed that 1 727 genes were divided into six co-expression modules. Notably, the brown and turquoise modules demonstrated significant correlations with X149 resistance. Within the brown and turquoise modules, GmNAC030 in turquoise module, a NAC transcription factor, showed the highest connectivity and was identified as a core transcription factor. Other NAC transcription factor genes in the turquoise module expression were similar to GmNAC030 included GmNAC043, GmNAC085, GmNAC092, and GmNAC101. Prediction of interacting protein indicated that the interacting proteins of GmNAC030, GmNAC043, and GmNAC092 were primarily transcription factors. Specifically, only the interacting proteins Glyma.02g131700 (bZIP1), Glyma.16g164800 (AP2-EREBP), and Glyma.08G118200 (WRKY48) were located within the module. RT-qPCR analysis demonstrated that GmNAC030 expression was predominantly induced by MeJA, while the other four NAC transcription factors were mainly induced by both MeJA and ETH. Conclusion The expression of five NAC transcription factors, GmNAC030, GmNAC043, GmNAC085, GmNAC092 and GmNAC101, is associated with soybean resistance to SMV and can be induced by MeJA and ETH.

Key words: soybean, soybean mosaic virus, WGCNA, NAC transcription factor, expression analysis

牛景萍, 赵婧, 郭茜, 王书宏, 赵晋忠, 杜维俊, 殷丛丛, 岳爱琴. 基于WGCNA鉴定大豆抗大豆花叶病毒NAC转录因子及其诱导表达分析[J]. 生物技术通报, 2025, 41(7): 95-105.

NIU Jing-ping, ZHAO Jing, GUO Qian, WANG Shu-hong, ZHAO Jin-zhong, DU Wei-jun, YIN Cong-cong, YUE Ai-qin. Identification and Induced Expression Analysis of Transcription Factors NAC in Soybean Resistance to Soybean Mosaic Virus Based on WGCNA[J]. Biotechnology Bulletin, 2025, 41(7): 95-105.

图/表 9

表1 RT-qPCR引物信息

Table 1 Information of primers for RT-qPCR

基因名称 Gene name	基因ID Gene ID	上游引物 Forward primer (5′-3′)	下游引物 Reverse primer (5′-3′)
GmNAC030	Glyma.05G195000	GGTTCAGTTCCAGCACGAGA	CGTGTCCATGTACAATTGGTCG
GmNAC043	Glyma.06G248900	TCTTCCCGCAAACACAACCT	TGACCCATTGCTGCATTCCA
GmNAC085	Glyma.12G149100	CGACATGCTGGAATCGTTGC	GTCCTGCTGCTGCATTGTTC
GmNAC092	Glyma.12G221500	ACAACTCGACGACGTTCTGG	CCCCCTTCACCCAAGTTCTG
GmNAC101	Glyma.13G279900	AACACGCTGCAACAACAACA	ATTCCCCGAAGCGAAATCGG
tublin		GGAGTTCACAGAGGCAGAG	CACTTACGCATCACATAGC

表2 转录因子统计

Table 2 Statistics of transcription factors (TF)

转录因子家族 TF family	基因数 Gene number	转录因子家族 TF family	基因数 Gene number
MYB	335	MIKC	84
ERF	305	Dof	79
bHLH	295	C3H	69
MYB_related	266	GATA	60
HB-other	204	ARF	55
WRKY	176	TCP	54
NAC	171	C2H2	53
M_type	144	HSF	52
bZIP	144	FAR1	49
B3	131	ZF-HD	47
GRAS	111	SBP	45
LBD	91	AP2	44
HD-ZIP	41	CPP	12
DBB	34	EIL	11
Nin-like	28	BBR-BPC	10
CO-like	22	GeBP	9
GRF	22	LSD	8
NF-YA	21	Whirly	7
SRS	21	HB-PHD	6
TALE	18	NF-X1	5
YABBY	17	RAV	4
BES1	15	S1Fa-like	4
CAMTA	15	LFY	2
E2F/DP	14	NZZ/SPL	1

图1 转录因子差异表达基因鉴定

Fig. 1 Identification of DEGs for transcription factors

图2 样本聚类图A：36个样本聚类图，3个红色的为离群样本；B：剔除离群样本后的33个样本聚类图

Fig. 2 Clustering diagram of samplesA: Clustering diagram of all 36 samples. The three outlier samples are written in red fonts. B: Clustering diagram of 33 samples after removing the three outlier samples

图3 加权基因共表达网络分析软阈值的确定和模块鉴定A：软阈值（β值）的确定；B：基因模块分类，树枝代表基因，不同颜色代表不同模块；C：模块与抗性表型相关热图，格子上方数字代表相关系数，下方数值是P值

Fig. 3 Determination of soft threshold power (β value) via WGCNA and identification of modulesA: Determination of soft threshold power (β value). B: Gene module classification, dendrogram indicate genes and different colors indicate different modules. C: Heat map of correlations between resistance trait and modules. The correlation coefficients are shown above each cell, and the P values are shown below

图4 抗病相关模块连通性前30个转录因子基因的可视化网络分析

Fig. 4 Visual network analysis of top 30 transcription factor genes in the disease resistance-related module connectivity

图5 NAC表达量热图和RT-qPCR验证A：5个NAC在X149和X97不同时间点的表达量热图；B：GmNAC030在X149中的RT-qPCR验证；*：P<0.05，**：P<0.01，下同

Fig. 5 Expression profiles of NAC and validation of NAC by RT-qPCRA: Expression profiles of five NAC at different time point X149 and X97. B: Validation of GmNAC030 in X149 by RT-qPCR; *: P<0.05, **: P<0.01,The same below

图6 NAC蛋白互作分析及互作蛋白在转录组中表达量热图A： Glyma.05g195000互作蛋白；B：Glyma.06G248900互作蛋白；C：Glyma.12G221500互作蛋白；D：存在于模块中的互作蛋白表达量热图

Fig. 6 Analysis of NAC protein interaction and expression profiles of interacting proteins in transcriptomeA: Interacting protein Glyma.05g195000. B: Interacting protein Glyma.06G248900. C: Interacting protein Glyma.12G221500. D: Expression profiles of interacting protein in the modules

图7 NAC转录因子受激素诱导表达分析

Fig. 7 Expression analysis of NAC transcription factors induced by hormone

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