基于4D Label-free技术研究AflaILVB/G/I基因对黄曲霉毒素合成的蛋白质组学分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0314

摘要/Abstract

摘要：

目的探究乙酰羟酸合成酶（AHAS）在黄曲霉毒素合成中的作用机制，特别是其通过代谢网络调控黄曲霉毒素合成的分子机理。方法采用4D Label-free蛋白质组学技术，系统地比较ΔAflaILVB/G/I菌株与野生型菌株的蛋白质表达谱差异，对获得的差异蛋白进行GO富集、KEGG富集和蛋白互作等分析。结果质谱分析结果显示，共鉴定出1 158个差异表达蛋白，其中521个表达上调，637个表达下调，与黄曲霉毒素合成相关的17个差异蛋白中，有14个明确属于黄曲霉毒素合成基因簇上的蛋白且均呈现显著性表达下调，该结果与RNA-Seq和RT-qPCR结果相对一致。将这17个蛋白与支链氨基酸合成相关的9个差异蛋白互作网络分析发现，AHAS对黄曲霉毒素合成的调控机制可能不是通过关键节点蛋白来实现，而是通过影响代谢通路的物质分配来实现的。结论 ΔAflaILVB/G/I菌株通过干扰缬氨酸和异亮氨酸的生物合成导致琥珀酰辅酶A和琥珀酸生成不足，进而引发一系列代谢重编程。这种代谢重编程不仅抑制黄曲霉的生长，还影响黄曲霉毒素的合成。

关键词: 乙酰羟酸合成酶, 支链氨基酸, 黄曲霉, 黄曲霉毒素, Label-free蛋白组学技术

Abstract:

Objective To investigate the role of acetohydroxyacid synthase (AHAS) in aflatoxin biosynthesis, particularly its molecular mechanism in regulating aflatoxin production through metabolic networks. Method A 4D Label-free proteomics approach was employed to systematically compare the protein expression profiles between theΔAflaILVB/G/I strain and the wild-type strain. Differentially expressed proteins (DEPs) were subjected to GO enrichment, KEGG pathway, and protein-protein interaction (PPI) network analyses. Result Mass spectrometry identified 1 158 DEPs, including 521 upregulated and 637 downregulated proteins. Among these, 17 were associated with aflatoxin biosynthesis, 14 of which were significantly downregulated and belonged to the aflatoxin biosynthetic gene cluster, consistent with RNA-Seq and RT-qPCR results. PPI network analysis of these 17 proteins and 9 DEPs related to branched-chain amino acid biosynthesis suggested that AHAS likely regulated aflatoxin production not through key hub proteins but by altering metabolic flux distribution. Conclusion TheΔAflaILVB/G/I strain disrupts valine and isoleucine biosynthesis, leading to insufficient succinyl-CoA and succinate levels. This metabolic reprogramming not only inhibits Aspergillus flavus growth but also suppresses aflatoxin synthesis.

Key words: acetohydroxyacid synthase, branched-chain amino acids, Aspergillus flavus, aflatoxin, Label-free proteomics technology

黄楚蓝, 曾睿, 陈佩榕, 赵亚荣, 王旭, 姚冬生. 基于4D Label-free技术研究AflaILVB/G/I基因对黄曲霉毒素合成的蛋白质组学分析[J]. 生物技术通报, 2025, 41(10): 321-333.

HUANG Chu-lan, ZENG Rui, CHEN Pei-rong, ZHAO Ya-rong, WANG Xu, YAO Dong-sheng. Proteomic Analysis Reveals the Role of AflaILVB/G/Ⅰ Gene in Aflatoxin Biosynthesis Based on 4D Label-free Technology[J]. Biotechnology Bulletin, 2025, 41(10): 321-333.

图/表 14

表1 本研究用到的曲霉菌株

Table 1 Aspergillus strains used in this study

菌株名称

Name of strain

基因型

Genotype

来源

Source

A. flavus NRRL 3357 TJES20.1

Δku70，ΔargB：： AfpyrG （Wild-type）

江苏师范大学杨教授惠赠

ΔAflaILVB/G/I（AFLA_000930）

Δku70，ΔargB：： AfpyrG，ΔAflaILVB/G/I：： argB

本研究构建

表2 荧光定量引物序列

Table 2 Primer sequences for fluorescence quantification

引物名称 Primer name	序列 Sequence（5'-3'）
Actin-QF	ACGGTGTCGTCACAAACTGG
Actin-QR	CGGTTGGACTTAGGGTTGATAG
aflP-QF	ACGAAGCCACTGGTAGAGGAGATG
aflP-QR	GTGAATGACGGCAGGCAGGT
aflO-QF	GATTGGGATGTGGTCATGCGATT
aflO-QR	GCCTGGGTCCGAAGAATGC
aflK-QF	CTCGCACTTTGGCATGTACG
aflK-QR	AATCCTCCCGCCTCAATCAC
aflD-QF	GTGGTGGTTGCCAATGCG
aflD-QR	CTGAAACAGTAGGACGGGAGC
hypC-QF	GCATGGTGCCTTACACATGG
hypC-QR	CCTACCAACCTCACGCTCTC

图1 质谱数据质量控制A：蛋白质谱总鉴定； B：特异性肽段的电荷和长度分布； C：定量蛋白的肽段数量分布； D：定量蛋白的相对分子质量分布

Fig. 1 Quality control of mass spectrometry dataA: Total protein identification by mass spectrometry. B: Charge and length distribution of specific peptides. C: Peptide number distribution of quantified proteins. D: Relative molecular weight distribution of quantified proteins

图2 样本重复性分析A：PCC分析热图； B：PCA主成分分析； C：RSD分析小提琴图； D：RSD分析箱线图

Fig. 2 Sample reproducibility analysisA: PCC analysis heatmap. B: PCA Principal component analysis. C:RSD Analysis violin plot. D: RSD analysis boxplot

图3 差异蛋白筛选A：火山图； B：聚类热图

Fig. 3 Differential protein screeningA: Volcano plot. B: Clustering heatmap

图4 差异蛋白GO功能富集分析A：上调的差异蛋白GO富集气泡图； B：下调的差异蛋白GO富集气泡图

Fig. 4 GO functional enrichment analysis of differentially expressed proteinsA: GO enrichment bubble plot of upregulated differentially expressed proteins. B: GO enrichment bubble plot of downregulated differentially expressed proteins

图5 差异蛋白KEGG功能富集分析A：上调的差异蛋白KEGG富集条形图； B：下调的差异蛋白KEGG富集条形图

Fig. 5 KEGG functional enrichment analysis of differentially expressed proteinsA: KEGG enrichment bar plot of upregulated differentially expressed proteins. B: KEGG enrichment bar plot of downregulated differentially expressed protein

图6 差异蛋白结构域富集分析

Fig. 6 Domain enrichment analysis of differentially expressed proteins

图7 亚细胞定位分析

Fig. 7 Subcellular localization analysis

表3 支链氨基酸合成途径涉及的差异蛋白

Table 3 Differentially expressed proteins involved in the branched-chain amino acid biosynthesis pathway

蛋白质编码 Protein accession	蛋白质描述 Protein description	基因名称 Gene name	P值 P value	调控类型Regulated type
B8MYP9	Branched-chain amino acid aminotransferase	AFLA_081880	2.204 49E-08	Down
B8N7W8	Dihydroxy acid dehydratase Ilv3， putative	AFLA_105610	4.045 78E-08	Up
B8N9W8	Acetohydroxy-acid synthase small subunit	AFLA_112640	4.260 63E-08	Up
B8N9L9	Threonine dehydratase	AFLA_111650	9.742 79E-08	Up
B8NEC1	Ketol-acid reductoisomerase， mitochondrial	AFLA_060300	1.855 62E-06	Up
B8N1U6	Pyruvate decarboxylase	AFLA_032500	2.141 48E-06	Up
B8N9Q5	L-serine dehydratase， putative	AFLA_112010	7.377 42E-05	Down
B8NEE4	Pyridoxal-phosphate dependent enzyme， putative	AFLA_060530	0.000 130 39	Down
B8NB44	Branched-chain-amino-acid aminotransferase	AFLA_044190	0.000 135 251	Up

表4 黄曲霉毒素合成途径涉及的差异蛋白

Table 4 Differentially expressed proteins involved in the aflatoxin biosynthesis pathway

蛋白质编码 Protein accession	蛋白质描述 Protein description	基因名称 Gene name	P值 P value	调控类型Regulated type
P55790	Sterigmatocystin 8-O-methyltransferase	omtA/aflP/AFLA_139210	1.205 77E-13	Down
Q9P900	Demethylsterigmatocystin 6-O-methyltransferase	omtB/aflO/AFLA_139220	1.503 66E-13	Down
B8NHY3	AflK/VERB synthase	vbs/aflK/AFLA_139190	1.187 86E-12	Down
B8NHZ4	AflM/dehydrogenase/ketoreductase	ver-1/aflM/AFLA_139300	6.337 76E-12	Down
B8NI02	AflD/reductase	nor-1/aflD/AFLA_139390	1.591 52E-11	Down
B8NHY1	AflW/monooxygenase	moxY/aflW/AFLA_139170	2.287 05E-11	Down
B8NHX9	AflY	hypA/aflY/AFLA_139150	3.178 44E-11	Down
B8N9Y6	Toxin biosynthesis ketoreductase	AFLA_112820	3.624 58E-11	Up
B8NHY0	AflX/monooxygenase/oxidase	ordB/aflX/AFLA_139160	2.447 17E-09	Down
B8NI03	Noranthrone monooxygenase	hypC/AFLA_139400	6.130 27E-09	Down
B8NT69	O-methyltransferase	AFLA_053520	3.688 78E-08	Down
B8NHY9	AflL/desaturase/ P450 monooxygenase	verb/aflL/AFLA_139250	5.666 24E-08	Down
B8NHZ6	AflJ/esterase	estA/aflJ/AFLA_139320	8.454 44E-08	Down
B8NHZ2	AflN/monooxygenase	ver-A/aflN/AFLA_139280	9.288 83E-08	Down
B8NI04	AflC/polyketide synthase	pskA/aflC/AFLA_139410	1.609 73E-05	Down
B8N0Z3	N-alkane-inducible cytochrome P450	AFLA_027680	0.000 725 794	Up
B8N628	O-methyltransferase family protein	AFLA_016120	0.017 023 299	Down

图8 差异蛋白互作网络分析椭圆形和正方形属于支链氨基酸合成途径差异蛋白互作到蛋白质，其中椭圆形与AFLA_000930蛋白有互作关系；三角形属于黄曲霉毒素合成途径差异蛋白互作到蛋白质；菱形为连接节点蛋白。红色代表上调的差异蛋白；蓝色代表下调的差异蛋白

Fig. 8 Protein-protein interaction network analysis of differentially expressed proteinsEllipses and squares indicate differentially expressed proteins in the branched-chain amino acid synthesis pathway that interact with proteins, where ellipses interact with the AFLA_000930 protein; triangles indicate differentially expressed proteins in the aflatoxin synthesis pathway that interact with proteins; diamonds indicate connecting node proteins. Red indicates upregulated differentially expressed proteins; blue indicates downregulated differentially expressed proteins

图9 黄曲霉毒素合成相关基因表达量测定

Fig. 9 Expression determination of aflatoxin biosynthesis-related genes

图10 细胞内代谢重编程分析×代表该途径受到AHAS功能缺陷干扰；红色代表上调的代谢途径；蓝色代表下调的代谢途径

Fig. 10 Analysis of intracellular metabolic reprogramming× indicates metabolic pathways affected by AHAS functional deficiency; red indicates upregulated metabolic pathways; blue indicates downregulated metabolic pathways

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