AOL-113转录因子对少孢节丛孢菌菌丝生长、胁迫响应及捕食能力的调控作用

doi:10.13560/j.cnki.biotech.bull.1985.2025-0603

摘要/Abstract

摘要：

目的为探究少孢节丛孢菌（Arthrobotrys oligospora）Zn（Ⅱ）₂Cys₆型转录因子AOL-113的生物学功能。方法利用同源重组的方法构建Δ AOL-113缺失株和CΔ AOL-113回补株，从菌丝生长特性、营养物质利用、环境胁迫响应、分生孢子产生及萌发和线虫捕食能力等表型进行分析，同时结合转录组学技术，在乙酸钠诱导条件下对WT菌株与Δ AOL-113缺失株进行差异表达基因分析，探究转录因子AOL-113的分子调控机制。结果转录因子AOL-113编码GAL4型锌指蛋白，与转录因子FacB有较高的同源性。与WT菌株相比，Δ AOL-113缺失株生长速率、碳源利用特别是脂肪酸的利用能力显著下降，对H₂O₂和甲萘醌这类氧化胁迫剂更敏感。此外，∆ AOL-113缺失株产生捕器数量及捕器形成速度显著下降，对线虫的降解消化利用能力也显著下降。回补株表型与WT株无显著差异。转录组分析证实，AOL-113缺失导致多个代谢通路（包括氧化磷酸化、脂质代谢、乙醛酸循环、过氧化物酶体、半乳糖代谢和糖酵解）中关键基因表达显著下调，从而影响菌丝生长、碳源利用、抗胁迫能力、捕食能力等多个生物学过程。结论转录因子AOL-113不仅通过调控细胞代谢（尤其是乙酸代谢和脂肪酸β氧化）为菌丝生长和捕食器形成提供能量支持，同时也参与调控捕食线虫和消化线虫的效率，为揭示少孢节丛孢菌从腐生向寄生生活方式转变的分子机制和家畜线虫病生防制剂的研发提供了新思路。

关键词: 少孢节丛孢菌, Zn₂（Ⅱ）Cys₆型转录因子, 碳源利用, 菌丝生长, 捕食能力

Abstract:

Objective To investigate the biological function of the Zn(II)₂Cys₆-type transcription factor AOL-113 of Arthrobotrys oligospora. Method The ΔAOL-113 deletion strain and the CΔAOL-113 complementary strain were constructed by homologous recombination, and the phenotypes such as mycelial growth, nutrient utilization, environmental adaptability, conidial production and germination, as well as nematode-trapping efficiency were subsequently analyzed. Additionally, transcriptome analysis was conducted to identify differentially expressed genes between the WT strain and the ΔAOL-113 deletion strain under sodium acetate conditions. Result The transcription factor AOL-113 gene encoded GAL4 zinc finger protein and shared high homology with the transcription factor FacB. Compared to the wild-type (WT) strain, the ∆AOL-113 strain presented significantly reduced growth rates and a marked decrease in carbon source utilization, especially fatty acid metabolism. It is more sensitive to oxidative stress agents such as H₂O₂ and menadione. The ∆AOL-113 strain also showed diminished trap production and a lower rate of sticky network formation, which significantly impaired its ability to degrade and digest nematodes. The complementary strain showed no significant phenotypic differences compared to the WT strain. Transcriptome analysis further revealed that the deletion of the AOL-113 led to the down-regulation of key genes involved in multiple pathways such as oxidative phosphorylation, lipid metabolism, glyoxylate cycle, peroxisome function, galactose metabolism, and glycolysis, this affected multiple biological processes such as mycelial growth, carbon source utilization, stress resistance, and predation ability. Conclusion The transcription factor AOL-113 not only provides energy support for hyphal growth and trap formation by regulating cellular metabolism (especially acetic acid metabolism and fatty acid β-oxidation), but also participates in regulating the efficiency of nematode predation and digestion, providing new ideas for revealing the molecular mechanism of the transition of A. oligosporus from a saprophytic to a parasitic lifestyle and the development of biocontrol agents for livestock nematode diseases.

Key words: Arthrobotrys oligospora, Zn(II)?Cys?-type transcription factor, carbon source utilization, mycelial growth, predatory ability

孙焱森, 魏立翔, 李若冰, 张程志, 聂宇航, 李杰, 才学鹏, 乔军, 孟庆玲. AOL-113转录因子对少孢节丛孢菌菌丝生长、胁迫响应及捕食能力的调控作用[J]. 生物技术通报, 2026, 42(2): 338-350.

SUN Yan-sen, WEI Li-xiang, LI Ruo-bing, ZHANG Cheng-zhi, NIE Yu-hang, LI Jie, CAI Xue-peng, QIAO Jun, MENG Qing-ling. Regulatory Roles of Transcription Factor AOL-113 in Mycelial Growth， Stress Response， and Predatory Ability of Arthrobotrys oligospora[J]. Biotechnology Bulletin, 2026, 42(2): 338-350.

图/表 9

表1 研究中使用的特异性引物

Table 1 Specific primers used in the study

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	长度 Length （bp）	基因目的 Gene description
AOL-113-F/AOL-113-R	ATGTCGTTGAAACGGTCG/GTCATTCCAGCTTGATAGAACCG	3 213	AOL-113基因全长
AOL-113-5F-pUC19	GTACTGAGAGTGCACCATATGCGACAGCCTTGGAGAGTTTGAC	1 861	AOL-113基因5′端基因序列
AOL-113-5R-HygR	GTTACTGAAATCTCCAACGGTTAGGAATATGTTCGTCAATGAGG	1 861	AOL-113基因5′端基因序列
HygR-F-113-5R	GACGAACATATTCCTAACCGTTGGAGATTTCAGTAACGTTAAG	1 441	潮霉素抗性基因
HygR-R-113-3F	CTACCCGGTTGACCAAGGTCGGAGACAGAAGATGATATTG	1 441	潮霉素抗性基因
AOL-113-3F-HygR	CATCTTCTGTCTCCGACCTTGGTCAACCGGGTAGAGG	1 865	AOL-113基因3′端基因序列
AOL-113-3R-pUC19	GACCATGATTACGCCAAGCTTTGCACACGTACTACAAAGCAAGC	1 865	AOL-113基因3′端基因序列
CΔ AOL-113-5F-pUC19	CTGAGAGTGCACCATATGCTTCCTCTTCAATTCCTCTTACAAGG	3 482	CΔ AOL-113基因回补全长及5′端侧翼序列
CΔ AOL-113-5R-NeoR	GTGCAGATTATTTGGATTGACAAGTATATCCTTCACCCATGACC	3 482	CΔ AOL-113基因回补全长及5′端侧翼序列
NeoR-F-113-5R	GTGAAGGATATACTTGTTCAATCCAAATAATCTGCACCG	919	G-418抗性基因
NeoR-R-113-3F	CCATCTATGTCACCTCTACCCCAGGTTGGGCGTCGCTTG	919	G-418抗性基因
CΔ AOL-113-3F-pUC19	CAAGCGACGCCCAACCTGGGGTAGAGGTGACATAGATGGAACG	1 382	CΔ AOL-113 3′端基因序列
CΔ AOL-113-3R-pUC19	GACCATGATTACGCCAAGCTTCAGTTGTGAAGAGCTTCTCCCTTG	1 382	CΔ AOL-113 3′端基因序列
Δ AOL-113YZ-F/Δ AOL-113YZ-R	GTATACTTACCAACCTGCTCC/ GCTACTTCCTGATGATATCCTTC	3 827/2 209	野生型和缺失转化子菌株的验证
CΔ AOL-113YZ-F/CΔ AOL-113YZ-R	CTTCCTCTTCAATTCCTCTTACAAGG/ GGTCATGGTGAAGGATATACTTGT	3 445/2 301	回补株转化子菌株的验证

图1 少孢节丛孢菌Zn（II）₂Cys₆型转录因子AOL-113的分子特征和系统发育分析A：转录因子AOL-113结构域特征。B：转录因子AOL-113同源基因的真菌进化树分析

Fig. 1 Molecular characteristics and phylogenetic analysis of the Zn2(II)Cys6-type transcription factor AOL-113 of A. oligosporaA: Domain characteristic of transcription factor AOL-113. B: Phylogenetic analysis of different fungi based on transcription factor AOL-113orthologs

图2 缺失株与回补株的构建与筛选A：缺失菌株电泳检测结果。M：DL-5000 DNA marker；1：阴性对照；2-3：缺失菌株阳性克隆。B：回补株电泳检测结果。M：DL-5000 DNA marker；1：回补菌株阳性克隆；2-4：阴性对照

Fig. 2 Construction and screening of deletion strains and complementary strainsA: Electrophoresis detection results of deletion strains. M: DL-5000 DNA marker; 1: negative control; 2-3: positive clones of deletion strains. B: Electrophoresis detection results of complementary strains. M: DL-5000 DNA marker; 1: positive clones of complementary strains; 2-4: negative control

图3 生长速率测定及菌丝细胞长度的比较A：TG、YPSSA、TYGA、PDA和CMY培养基上28 ℃培养5 d菌落形态观察及生长速率测定。B：菌丝细胞的CFW染色，比例尺=20 μm，菌丝隔膜用白色箭头表示

Fig. 3 Comparison of growth rate and mycelial cell lengthA: TG, YPSSA, TYGA, PDA and CMY media were incubated at 28 ℃ for 5 d for colony morphology observation and growth rate measurement. B: CFW staining of mycelia. Scale = 20 μm, the hyphal septa are denoted by white arrows. *P<0.05, **P<0.01. The same below

图4 不同碳、氮源和脂肪酸利用能力分析

Fig. 4 Analysis of utilization capacity of different carbon, nitrogen sources and fatty acid

图5 对高渗溶液、细胞壁合成抑制剂、氧化剂、热胁迫和pH剂的敏感性

Fig. 5 Sensitivities to osmotic agents, cell wall-perturbing agents, oxidants agents, thermal agents and pH agents

图6 产孢率、孢子萌发率的比较及分生孢子的荧光观察A：分生孢子梗侧拍观察。B：产孢率测定。C：孢子萌发测定。D：孢子萌发观察。E：分生孢子荧光观察及孢子长度测定

Fig. 6 Comparison of sporulation rate, spore germination rate and fluorescence observation of conidiaA: Side view of the phloem peduncle. B: Determination of sporulation rate. C: Spore germination rate. D: Observation of conidial germination. E: Fluorescence observation of conidia and spore length determination

图7 捕器形成和捕食能力比较A：不同时间点捕器形成情况及捕器计数。B：线虫捕食情况的观察及捕食率测定

Fig. 7 Comparison of the trap formation and predatory abilityA: Trap formation and traps counts at different time points. B: Observations of nematode predation and determination of predation rate

图8 乙酸钠诱导条件下少孢节丛孢菌WT株和Δ AOL-113缺失株DEGs分析A：DEGs火山图。上调DEGs为红色；下调的DEGs为蓝色。B：下调DEGs的GO富集分析。C：下调DEGs的KEGG富集分析。D：差异表达基因RT-qPCR验证

Fig. 8 Analysis of DEGs between WT and Δ AOL-113 of Arthrobotrys oligospora exposed to sodium acetate (SA)A: Volcano plot of DEGs. Up-regulated DEGs in red; down-regulated DEGs in blue. B: GO enrichment analysis of downregulated DEGs. C: KEGG enrichment analysis of downregulated DEGs. D: Validation of transcriptomic sequencing using RT-qPCR

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