基于WGCNA挖掘野生大豆耐镉关键基因

doi:10.13560/j.cnki.biotech.bull.1985.2024-1113

摘要/Abstract

摘要：

目的野生大豆具有耐逆境特性，逐渐成为改良栽培大豆的种质资源，明确野生大豆耐镉分子调控机制，为培育耐性大豆品种提供依据。方法以冀东地区200份野生大豆为试验材料，利用含有75 mol/L CdCl₂的Hoagland营养液处理野生大豆幼苗，并测定幼苗干重，分别对镉处理24和48 h的耐镉野生大豆R、敏感材料S进行转录组测序，对差异表达基因进行KEGG和GO富集分析，利用加权基因共表达网络分析挖掘耐镉核心基因。结果 200份野生大豆幼苗受到不同程度镉的胁迫，与对照相比，镉处理条件下，幼苗地上部干重、地下部干重明显下降。转录组分析结果显示，在R和S材料中分别鉴定到6 443和4 496个差异表达基因。经GO和KEGG分析，发现这些差异表达基因富集到光合作用、逆境响应途径。结合加权基因共表达网络分析，挖掘到参与调控野生大豆耐镉的turquoise和blue关键模块与野生大豆耐镉性状显著相关。根据模块内基因的连接度和基因功能注释，预测LOC114376469、LOC114412091、LOC114388638、LOC114399512等8个基因可能在野生大豆镉胁迫过程中发挥作用。结论鉴定了2个与野生大豆耐镉相关的特异性模块，筛选到LOC114376469、LOC114412091、LOC114388638、LOC114399512等与野生大豆耐镉相关的核心基因。

关键词: 野生大豆, 镉胁迫, WGCNA, 核心基因

Abstract:

Objective Wild soybean has characteristics of tolerance to stress, gradually becoming a germplasm resource for improved cultivated soybeans. It is significant to clarify the molecular regulation mechanism of tolerance to cadmium in wild soybeans and provide a basis for cultivating tolerant soybean varieties. Method In this study, 200 wild soybeans in eastern Hebei were used as experimental materials. Wild soybean seedlings were treated with Hoagland nutrient solution containing 75 mol/L CdCl₂, and the dry weight of the seedlings was measured. Transcriptome sequencing was performed on Cd-tolerant wild soybean R and sensitive material S at 24 h and 48 h under Cd treatment, respectively. KEGG and GO enrichment analyses were performed on differentially expressed genes (DEGs), and weighted gene co-expression network analysis (WGCNA) was used to detect Cd-tolerant core genes. Result The 200 wild soybean seedlings have shown remarkable differences under stress. Compared with the control, the shoot dry weight and root dry weight of seedlings under cadmium treatment significantly reduced. Transcriptome analysis showed that 6 443 and 4 496 DEGs were identified in R and S materials, respectively. GO and KEGG analyses found that these DEGs were enriched in photosynthesis and stress response pathways. Combined with WGCNA, the key turquoise and blue modules were significantly correlated with cadmium tolerance in wild soybeans. According to the connectivity and functional annotation of the genes in the module, it was predicted that 8 genes, such as LOC114376469, LOC114412091, LOC114388638, and LOC114399512 may play a role in the process of cadmium stress in wild soybean. Conclusion Two specific modules related to the tolerance of wild soybean to cadmium are identified, and LOC114376469, LOC114412091, LOC114388638, and LOC114399512 are screened to be the core genes related to the tolerance of wild soybean to cadmium.

Key words: wild soybean, Cd stress, WGCNA, core gene

朱丽娟, 张锴, 温晓蕾, 褚佳豪, 史凤玉, 王艳丽. 基于WGCNA挖掘野生大豆耐镉关键基因[J]. 生物技术通报, 2025, 41(8): 124-136.

ZHU Li-juan, ZHANG Kai, WEN Xiao-lei, CHU Jia-hao, SHI Feng-yu, WANG Yan-li. Mining the Core Genes Being Tolerant to Cadmium in Wild Soybean by WGCNA[J]. Biotechnology Bulletin, 2025, 41(8): 124-136.

图/表 11

表1 镉胁迫下野生大豆幼苗性状描述性分析

Table 1 Description analysis of wild soybean seedlings under Cd treatment

性状 Trait	全距 Range	最小值 Min	最大值 Max	平均值 Average	标准差 Standard deviation
SDW-CK1 （g）	0.090	0.006	0.096	0.017	0.008
SDW-Cd1 （g）	0.049	0.001	0.050	0.013	0.005
RDW-CK1 （g）	0.043	0.002	0.045	0.007	0.005
RDW-Cd1 （g）	0.029	0.001	0.030	0.005	0.003
RSR-CK1	1.265	0.149	1.414	0.373	0.151
RSR-Cd1	2.037	0.077	2.114	0.404	0.225
CTC-SDW1	1.643	0.199	1.842	0.802	0.212
CTC-RDW1	2.363	0.146	2.509	0.848	0.315
CTC-RSR1	3.332	0.165	3.497	1.122	0.467
SDW-CK2 （g）	0.082	0.007	0.089	0.016	0.008
SDW-Cd2 （g）	0.059	0.002	0.061	0.014	0.007
RDW-CK2 （g）	0.056	0.002	0.058	0.006	0.005
RDW-Cd2 （g）	0.031	0.001	0.032	0.005	0.003
RSR-CK2	1.411	0.158	1.569	0.364	0.167
RSR-Cd2	1.180	0.062	1.242	0.365	0.165
CTC-SDW2	3.570	0.166	3.736	0.877	0.438
CTC-RDW2	2.500	0.014	2.514	0.856	0.342
CTC-RSR2	2.824	0.039	2.863	1.068	0.428
D1	1.05	0.14	1.19	0.459	0.147
D2	1.14	0.02	1.16	0.512	0.181

图1 两次重复下野生大豆幼苗干重相关性分析*、**和***分别表示在0.05、0.01和0.001水平上差异显著

Fig. 1 Correlation analysis of dry weight of wild soybean seedlings under two repetitions*, ** and *** indicate extremely significant correlaton at 0.05, 0.01 and 0.001 levels, respectively

图2 耐镉和敏感野生大豆的幼苗

Fig. 2 Cadmium tolerant, sensitive wild soybean seedlings

图3 转录组测序数据分析A：主成分分析；B：相关性检验；C：差异表达基因热图。R0、R24、R48、S0、S24、S48代表R和S材料镉胁迫0、24和48 h。下同

Fig. 3 Analysis of transcriptome sequencing dataA: PCA analysis. B: Correlation test among samples. C: Heat map of DEGs. R0, R24, R48, S0, S24, and S48 indicate 0, 24, and 48 h under Cd stress in R and S. The same below

图4 镉胁迫下野生大豆R和S差异表达基因分析A：R和S材料不同时间点差异表达基因数量；B：R在不同时间点差异表达基因韦恩图；C：S在不同时间点差异表达基因韦恩图；D：R和S材料胁迫24 h差异表达基因韦恩图；E：R材料和S材料胁迫48 h差异表达基因韦恩图

Fig. 4 Analysis of DEGs of R and S in wild soybean under cadmium stressA: Number of DEGs. B: Venn diagram of DEGs in R at different time. C: Venn diagram of DEGs in S at different time. D: Venn diagram of DEGs in R and S at 24 h under treatment. E: Venn diagram of DEGs in R and S at 48 h under treatment

图5 GO富集分析A、B：R材料在胁迫24 h（A）、48 h（B）的差异表达基因GO富集分析；C、D：S材料在胁迫24 h（C）、48 h（D）的差异表达基因GO富集分析

Fig. 5 GO enrichment analysisA, B: GO enrichment analysis of DEGs in R at 24 h (A) and 48 h (B). C, D: GO enrichment analysis of DEGs in S at 24 h (C) and 48 h (D)

图6 KEGG富集分析A、B：R材料在胁迫24 h（A）和48 h（B）差异表达基因KEGG富集分析；C、D：S材料在胁迫24 h（C）和48 h（D）差异表达基因KEGG富集分析

Fig. 6 KEGG enrichment analysiA, B: KEGG enrichment analysis of DEGs in R at 24 h (A) and 48 h (B) under stress. C, D: KEGG enrichment analysis of DEGs in S at 24 h (C) and 48 h (D) under stress

图7 镉胁迫下野生大豆差异表达基因的WGCNA分析A：共表达模块划分；B：表型与基因模块相关性；C：blue模块共表达调控网络；D：turquoise模块共表达调控网络

Fig. 7 WGCNA enrichment analysis of DEGs in wild soybean under Cd stressA: Co-expression module division. B: Gene module eigenvalue connectivity heat map. C: Blue module co-expresses the regulatory network. D: Turquoise module co-expresses the regulatory network

表2 八个WGCNA关键基因的功能注释

Table 2 Functional annotation of 8 WGCNA hub genes

关键基因 Gene ID	基因功能注释 Gene function	同源基因 Orthologs
LOC114375809	Glutamine synthetase leaf isozyme， chloroplastic-like	Glyma 13G210800v4
LOC114376496	Protein DETOXIFICATION 42-like	Glyma 13G339800v4
LOC114377985	Stromal 70 kD heat shock-related protein， chloroplastic-like	Glyma 12G166200v4
LOC114387053	Phosphoglycerate kinase， cytosolic-like	Glyma 15G261900v4
LOC114388638	Heat shock 70 kD protein 14-like	Glyma 15G014400v4
LOC114399512	Magnesium-chelatase subunit ChlH， chloroplastic-like	Glyma 19G139300v4
LOC114406715	Magnesium-chelatase subunit ChlH， chloroplastic-like	Glyma 03G137000v4
LOC114412091	FAM10 family protein At4g22670-like	Glyma 05G051600v4

图8 野生大豆耐镉候选基因RT-qPCR分析

Fig. 8 RT-qPCR analysis of candidate genes for cadmium tolerance in wild soybean

图9 野生大豆耐镉核心基因编码蛋白互作蛋白的预测

Fig. 9 Prediction of protein interaction proteins encoded by cadmium-tolerant hub gene in wild soybean

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