Mycosystema

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The role of C₂H₂-type transcription factor AflAZF1 in the growth, development and aflatoxin synthesis regulation of Aspergillus flavus

LIN Hongyi, LI Mingzhi, GUO Huihua, YUAN Jun, WANG Shihua, WANG Xiuna

Mycosystema, 2026, 45(2): 250176. DOI: 10.13346/j.mycosystema.250176

引物名称 Primer	引物序列 Oligonucleotide sequence (5′→3′)	目的 Use
pyrG/F	GCCAGTACGAGTGTTGTGGAG	扩增pyrG基因片段 Amplification of pyrG gene fragments
pyrG/R	GTCAGACACAGAATAACTCTC	扩增pyrG基因片段 Amplification of pyrG gene fragments
054800 5F/F	GGTATGGACTGGCATGGATAC	融合 PCR 正向引物 Fusion PCR forward primers
054800 3F/R	GTAATATCAAGACTGCGACGCG	融合 PCR 反向引物 Fusion PCR reverse primers
054800 5F/EF	CATTGAGGAACGATGCCATTAC	扩增AflAZF1上游同源臂片段 Amplification of AflAZF1 upstream homology arm fragment
054800 5F/R	GGGTGAAGAGCATTGTTTGAGGCGTCTTTGCTATCTACTCGTCTC
054800 3F/ER	CTTCAATTGATCGTGTTGACAC	扩增AflAZF1下游同源臂片段 Amplification of AflAZF1 downstream homology arm fragments
054800 3F/F	GCATCAGTGCCTCCTCTCAGACGCCGTACTGGTCTTCTGTTCG
pyrG/TR	GTCTGAGAGGAGGCACTGATGC	ΔAflAZF1验证引物 ΔAflAZF1 validation primers
pyrG/TF	GCCTCAAACAATGCTCTTCACCC	ΔAflAZF1验证引物 ΔAflAZF1 validation primers
054800 RT/F	GCCATTCACCTGTCTGTTGGAC	AflAZF1-ORF验证引物 AflAZF1-ORF validation primers
054800 RT/R	CCTACGATCCTTTCCACGAC	AflAZF1-ORF验证引物 AflAZF1-ORF validation primers

Table 2 The primers used in this study

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Table 1 The strains used in this study
Fig. 1 The phylogenetic tree and domain analysis of the AZF1 proteins in Aspergillus flavus and other fungi. A: The phylogenetic tree of AZF1 proteins. The phylogenetic tree were constructed using MEGA7.0 software. The protein information is as follow: AflAZF1 (XP_002378224.1, A. flavus NRRL3357); AnAZF1 (A5ABV9.1, A. niger CBS 513.88); AfAZF1 (EDP47468.1, A. fumigatus A1163); TrAZF1 (XP_006961893.1, Trichoderma reesei QM6a); Azf1p (NP_014756.3, Saccharomyces cerevisiae S288C); CON1 (ABB89847.1, Pyricularia grisea); ApAZF1 (KJK64458.1, A. parasiticus); AoAZF1 (EIT80398.1, A. oryzae); AnAZF1 (XP_664107.1, A. nidulans); FgAZF1 (WXC62894.1, Fusarium graminearum); AtAZF1(GHJ86605.1, Arabidopsis thaliana). B: The domain of AZF1 proteins.
Fig. 2 Construction of AflAZF1 deletion mutant strain of Aspergillus flavus. A: Strategy of the construction of the AflAZF1 knockout mutant. B: PCR verification of WT (wild type) and ΔAflAZF1 strains. Knockout transformants are represented by T1, T2, T3, and T4; WT strain is the positive control; CK uses ddH₂O as a template, serving as the negative control; Gel wells numbered P1, P2, and P3 represent the amplification results using primers P1, P2, and P3, respectively. Marker: DL2000.
Fig. 3 Effects of AflAZF1 in Aspergillus flavus on growth and conidial formation. A: Growth and conidial formation of the WT and ΔAflAZF1 strains. B: Statistical analysis of the colony diameter of WT and ΔAflAZF1 strains. Relative growth=colony diameter/wild-type colony diameter. C: Statistical analysis of the conidia produced by WT and ΔAflAZF1. Error bars represent the standard deviation of five replicates. * Indicates a significant difference between the WT and ΔAflAZF1 at 0.05 level; ** Indicate an extremely significant difference between the two strains at 0.01 level. The same below.
Fig. 4 Deletion of the AflAZF1 gene results in the inability of Aspergillus flavus to form sclerotia. A: Sclerotia production by WT and ΔAflAZF1 on YEPS culture medium. B: Statistical analysis of sclerotial yield of WT and ΔAflAZF1 on the 7th day. ND indicates that no sclerotia were detected.
Fig. 5 AflAZF1 regulates the carbon source utilisation process in Aspergillus flavus. A: Growth of WT and ΔAflAZF1 on different carbon source media. B: Statistics analysis of colony diameters of WT and ΔAflAZF1 on eight types of carbon source media on day 5. C: Statistical analysis of conidia produced by WT and ΔAflAZF1.
Fig. 6 AflAZF1 mediates Aspergillus flavus adaptation to different acidic and alkaline environments. A: Growth of WT and ΔAflAZF1 on different pH media. B: Statistical analysis of the colony diameter of WT and ΔAflAFZ1. C: Statistical analysis of the conidia produced by WT and ΔAflAZF1.
Fig. 7 AflAZF1 negatively regulates the AFB₁ biosynthesis. A: TLC analysis of the production of AFB₁ by WT and ΔAflAFZ1 on a YES medium. AFB₁ is the standard aflatoxin. B: Quantitative analysis of the yield of AFB₁ produced by WT and ΔAflAFZ1 on YES medium. Concentration of standard AFB₁ is adjusted to 1 μg/mL, then the relative quantification of AFB₁ was analyzed using GeneTools software. The same below.
Fig. 8 Effects of AflAZF1 on the pathogenicity of Aspergillus flavus. A: Growth of WT and ΔAflAZF1 strains on peanut. B: Quantitative analysis of the conidia produced by WT and ΔAflAZF1 on peanuts. C: TLC analysis of AFB₁ from WT and ΔAflAZF1. D: Quantitative analysis of AFB₁ produced by WT and ΔAflAZF1.

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