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Characterization of the promoter of the nitrate transporter-encoding gene nrtA in Aspergillus nidulans

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Abstract

Aspergillus nidulans nrtA encodes a nitrate transporter that plays an important role in the \({\text{NO}}_{{3}}^{ - }\) assimilatory process. Many studies have focused on protein functions rather than gene regulation. The knowledge of nrtA\({\text{NO}}_{{3}}^{ - }\) uptake process, particularly in the regulation mechanism of transcription factors AreA and NirA on nrtA transcription, is very limited. Herein, we investigated the transcriptional regulation of nrtA in response to various N-sources in detail and characterized the promoter activity of nrtA. We confirmed that nrtA was induced by \({\text{NO}}_{{3}}^{ - }\) and repressed by preferred N-sources. Additionally, for the first time, we found that the transcription of nrtA increased under N-starvation conditions. AreA mediates nrtA transcription under both \({\text{NO}}_{{3}}^{ - }\) and N-starvation conditions, while NirA is effective only under \({\text{NO}}_{{3}}^{ - }\) conditions. All of the proposed AreA and NirA binding sites in the promoter region were capable of binding to their corresponding transcription factors in vitro. In vivo, all of the NirA binding sites showed regulation activities, but to AreA, only several of the initiation-codon-proximal binding sites participated in nrtA transcription. Moreover, the active binding sites contributed in different degrees of regulation strength to nrtA transcription, which is unrelated to the distance between the binding sites and initiation codon. These results provided an extensive map of nrtA promoter, defining the functional regulatory elements of A. nidulans nrtA.

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Fig. 1
Fig. 2

source for 14 h. The resulting mycelia were separately treated with the indicated N-sources (10 mM) for another 3 h. GUS activity of the strains A. n. PnrtA as WT (black column), areA mutant areAΔ (white column) and nirA mutant nirAΔ (gray column) were analyzed. GUS activity in 14 h precultivated strains was set as the control (initial GUS). The GUS activity of nrtA constitutively expression strains (WT-nrtAC, black stripe; areAΔ-nrtAC, white stripe) were analysed by the same condition described above, shown as an insert. Values are the mean ± standard deviation (SD) of at least three independent experiments. **p < 0.01; ***p < 0.001 vs. the untreated group (0 min) of WT strain. ##p < 0.01 vs. the untreated group (0 min) of nirAΔ strain. The marked p values in the figure are the data compared between the WT and nirAΔ in each nitrogen induction condition

Fig. 3
Fig. 4

for 14 h. After that, the resulting strains were divided into three parts, one was collected for the activity assay and used as a control (untreated, gray column). The other two parts were treated with 10 mM nitrate (red column) or ammonium (green column) as the sole N-source for 3 h. The positions of the proposed AreA and NirA binding sites are shown as indicated. The arrow direction represents the binding site on the coding strand or antisense strand of the uidA reporter gene. a Deletion analysis of the nrtA promoter. The ATG indicates the initiation codon of the uidA gene, and the numbers given for each deletion mutant show the distance in base pairs from the initiation codon to the deletion endpoint. b Transcriptional factor binding site mutation analysis. The data are presented as the average of three independent experiments, and the bars indicate standard deviation. N.S., not significant; *p < 0.05; **p < 0.01; ****p < 0.0001

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Funding

This study was funded by National Major Science and Technology Projects of China (2019ZX09739-001), National Natural Science Foundation of China (21672065 and 21636003), the Fundamental Research Funds for the Central Universities (22221818014), the 111 Project (B18022), the Project Funded by the International S&T Innovation Cooperation Key Project (2017YFE0129600).

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Correspondence to Shengmin Zhou.

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We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

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Communicated by Stefan Hohmann.

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Liu, Y., Li, H., Li, J. et al. Characterization of the promoter of the nitrate transporter-encoding gene nrtA in Aspergillus nidulans. Mol Genet Genomics 295, 1269–1279 (2020). https://doi.org/10.1007/s00438-020-01700-x

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