Biotechnology Bulletin ›› 2025, Vol. 41 ›› Issue (10): 321-333.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0314
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HUANG Chu-lan1(
), ZENG Rui2, CHEN Pei-rong2, ZHAO Ya-rong2(
), WANG Xu2, YAO Dong-sheng1(
)
Received:2025-03-26
Online:2025-10-26
Published:2025-10-28
Contact:
ZHAO Ya-rong, YAO Dong-sheng
E-mail:1515817278@qq.com;zyr520zyr@163.com;dsyao2001@263.net
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.
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 |
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 |
| 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 |
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 |
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
Fig. 2 Sample reproducibility analysisA: PCC analysis heatmap. B: PCA Principal component analysis. C:RSD Analysis violin plot. D: RSD analysis boxplot
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
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
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 |
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 |
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 |
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 |
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
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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