大豆转录因子GmWRKY23a对有益绿针假单胞菌根系定殖的调控

doi:10.13560/j.cnki.biotech.bull.1985.2025-1356

摘要/Abstract

摘要：

目的明确大豆转录因子GmWRKY23a在调控有益绿针假单胞菌（Pseudomonas chlororaphis）IRHB3根系定殖过程中的作用机制。方法以大豆品种‘南豆12’（Glycine max cv. Nandou 12）为材料，结合生物信息学、分子生物学分析及大豆毛状根瞬时遗传转化技术，系统研究GmWRKY23基因及其编码蛋白的结构特征，解析GmWRKY23a在IRHB3根系定殖中的应答调控机制。结果从NCBI和Phytozome数据库共检索到4个标注为大豆GmWRKY23的基因序列，其编码产物均含有典型的WRKY结构域，定位于细胞核，但在蛋白结构和启动子顺式作用元件组成方面存在差异。其中，GmWRKY23a呈现明显的组织特异性表达，并可被IRHB3、病原菌尖孢镰刀菌（Fusarium oxysporum）B3S1、激素（SA、BR、MeJA）以及盐胁迫（NaCl）显著诱导。功能分析表明，过表达GmWRKY23a可有效促进IRHB3在大豆根系的定殖；该转录因子正调控异黄酮合成关键酶基因（CHS8、CHR1、IFS1）的表达，并能解除IRHB3定殖对IFS1的转录抑制，协同增强CHS8和CHR1的表达水平。结论 GmWRKY23a通过激活异黄酮合成途径相关基因转录，调控大豆对有益绿针假单胞菌的根系定殖过程，为揭示植物与有益微生物互作中转录调控与次生代谢协同机制提供了新的理论依据。

关键词: GmWRKY23a, 绿针假单胞菌IRHB3, 根际定殖, 植物免疫, 异黄酮合成

Abstract:

Objective This study aims to elucidate the role of the soybean transcription factor GmWRKY23a in regulating root colonization by the beneficial bacterium Pseudomonas chlororaphis IRHB3. Methods Using the soybean cultivar Glycine max cv. Nandou 12, we systematically investigated the structural features of the GmWRKY23 gene and its encoded protein through a combination of bioinformatic analysis, molecular biology approaches, and the soybean hairy root-based transient genetic transformation. The regulatory mechanism of GmWRKY23a in response to IRHB3 root colonization was further characterized. Results Four gene sequences annotated as GmWRKY23 were retrieved from the NCBI and Phytozome databases. All encoded proteins contained the conserved WRKY domain and localized in the nucleus, but exhibited variations in protein structure and promoter cis-acting element composition. Among them, GmWRKY23a showed distinct tissue-specific expression and was significantly induced by IRHB3, the pathogen Fusarium oxysporum B3S1, phytohormones (SA, BR, MeJA), and salt stress (NaCl). Functional assays revealed that overexpression of GmWRKY23a promoted IRHB3 colonization in soybean roots. GmWRKY23a positively regulated the expression of key isoflavone biosynthetic genes, including chalcone synthase (CHS8), chalcone reductase (CHR1), and isoflavone synthase (IFS1), alleviated the transcriptional repression of IFS1 caused by IRHB3 colonization, and synergistically enhanced the expression of CHS8 and CHR1 in coordination with IRHB3. Conclusion GmWRKY23a positively modulates root colonization by the beneficial P. chlororaphis viatranscriptional activation of the isoflavone biosynthesis pathway. These findings provide new insights into the coordinated regulation of transcription and secondary metabolism in plant-beneficial microbe interactions.

Key words: GmWRKY23a, Pseudomonas chlororaphis IRHB3, root colonization, plant immunity, isoflavone biosynthesis

徐焱, 陈玲, 黄灵琳, 常小丽. 大豆转录因子GmWRKY23a对有益绿针假单胞菌根系定殖的调控[J]. 生物技术通报, 2026, 42(8): 287-297.

XU Yan, CHEN Ling, HUANG Ling-lin, CHANG Xiao-li. Regulation of Root Colonization by the Beneficial Bacterium Pseudomonas chlororaphis via Soybean Transcription Factor GmWRKY23a[J]. Biotechnology Bulletin, 2026, 42(8): 287-297.

图/表 8

图1 大豆GmWRKY23氨基酸系统进化树基于大豆与其他物种WRKY23的氨基酸序列构建系统发育树，包括大豆（Glycine max）、豇豆（Vigna unguiculata）、绿豆（Vigna radiata var. radiata）、木豆（Cajanus cajan）、小豆（Vigna angularis）、野大豆（Glycine soja）、花生（Arachis hypogaea）、拟南芥（Arabidopsis thaliana）、烟草（Nicotiana tabacum）及水稻（Oryza sativa）

Fig. 1 Phylogenetic tree based on amino acid sequences of soybean GmWRKY23The phylogenetic tree was constructed based on the amino acid sequences of WRKY23 from soybean and other species, including soybean (Glycine max), cowpea (Vigna unguiculata), mung bean (Vigna radiata var. radiata), pigeon pea (Cajanus cajan), adzuki bean (Vigna angularis), wild soybean (Glycine soja), peanut (Arachis hypogaea), Arabidopsis thaliana, tobacco (Nicotiana tabacum), and rice (Oryza sativa)

图2 大豆GmWRKY23基因结构、保守结构域及基序分析A：GmWRKY23基因结构；B：GmWRKY23保守结构域；C：GmWRKY23基序对比

Fig. 2 Analysis of gene structures, conserved domains and motifs of soybean GmWRKY23 genesA: Gene structure of GmWRKY23; B: Conserved structures of GmWRKY23; C: Motif of GmWRKY23 genes

图3 大豆GmWRKY23启动子顺式作用元件分析

Fig. 3 Analysis of cis-acting elements in the promoters of soybean GmWRKY23 genes

图4 四个大豆GmWRKY23的蛋白序列比对* 代表间隔10个氨基酸

Fig. 4 Sequence alignment of four soybean GmWRKY23 proteins* indicates an interval of 10 amino acids

图5 GmWRKY23a蛋白在烟草叶片亚细胞定位Bright为明场观察结果；eYFP为黄色荧光蛋白标签，mCherry为红色核定位标记信号，Merge为叠加场，绿色荧光与红色荧光完全重叠呈现橙色。p1300-YFP为空载对照，GmWRKY23a-YFP为GmWRKY23a与YFP的融合表达蛋白

Fig. 5 Subcellular localization ofGmWRKY23a in Nicotiana tabacum leavesBright: bright field; eYFP: enhanced yellow fluorescent protein tag; mCherry: red fluorescent nuclear localization marker; Merge: merged field, where complete overlap of green and red fluorescence appears as orange. p1300-YFP: empty vector control; GmWRKY23a-YFP: fusion protein of GmWRKY23a and YFP

图6 大豆GmWRKY23a基因的表达模式A：组织特异性表达；B：生物胁迫处理后GmWRKY23a相对表达；C：非生物胁迫处理后GmWRKY23a相对表达。图中数据以平均值±标准差（Mean ± SD）表示。组间差异采用单因素方差分析和双因素方差分析检验，不同字母代表存在显著差异（P<0.05）

Fig. 6 Expression pattern of GmWRKY23a in soybeanA: Tissue-specific expression. B: Relative expression of GmWRKY23a under biotic stress treatment. C: Relative expression of GmWRKY23a under abiotic stress treatment. Data are presented as mean ± standard deviation (Mean ± SD). Differences between groups were analyzed using one-way ANOVA or two-way ANOVA, and different lowercase letters indicate statistically significant differences (P<0.05)

图7 GmWRKY23a过表达大豆毛状根的构建和IRHB3在毛状根中的定殖量A：转入对照质粒载体pCAMBIA1300-GFP（p1300）和过表达载体pCAMBIA1300-GmWRKY23a-GFP（OE-GmWRKY23a）的大豆毛状根；B：对照和过表达毛状根中GmWRKY23a的表达量；C：对照和过表达毛状根中IRHB3的定殖量。B中组间差异采用t检验，不同字母代表存在显著差异（P<0.05）

Fig. 7 Construction of soybean hairy roots overexpressing GmWRKY23a and colonization abundance of IRHB3 in hairy rootsA: Soybean hair roots carrying control vector pCAMBIA1300-GFP (p1300) and the overexpression vector pCAMBIA1300-GmWRKY23a-GFP (OE-GmWRKY23a); B: expression levels of GmWRKY23a in the control (p1300) and overexpressed (OE-GmWRKY23a) hairy roots; C: colonization abundance of IRHB3 in control and overexpressing hairy roots. Statistical analysis in Figure B was performed using the t-test, and different lowercase letters indicate statistically significant differences (P<0.05)

图8 异黄酮合成及代谢通路相关基因的表达结果组间差异采用单因素方差分析，不同字母代表显著差异（P<0.05）

Fig. 8 Expression of genes related to isoflavone biosynthesis and metabolism pathwayDifferences between groups were tested by one-way ANOVA. Different lowercase letters indicate significant differences at P<0.05

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