Liu He, Qiao Jinjin, Shangguan Jiaolei, Guo Xiaoyu, Xing Zhenzhen, Zhou Xiaolin, Zhao Mingwen, Zhu Jing
Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China.
J Fungi (Basel). 2023 Apr 26;9(5):516. doi: 10.3390/jof9050516.
Fungal AreA is a key nitrogen metabolism transcription factor in nitrogen metabolism repression (NMR). Studies have shown that there are different ways to regulate AreA activity in yeast and filamentous ascomycetes, but in , how AreA is regulated is unknown. Here, a gene from with similarity to of filamentous ascomycetes was identified. The NmrA interacted with the C-terminal of AreA according to yeast two-hybrid assay. In order to determine the effect of NmrA on the AreA, 2 silenced strains of , with silencing efficiencies of 76% and 78%, were constructed using an RNA interference method. Silencing resulted in a decreased content of AreA. The content of AreA in i-3 and i-48 decreased by approximately 68% and 60%, respectively, compared with that in the WT in the ammonium condition. Under the nitrate culture condition, silencing resulted in a 40% decrease compared with the WT. Silencing also reduced the stability of the AreA protein. When the mycelia were treated with cycloheximide for 6 h, the AreA protein was almost undetectable in the silenced strains, while there was still approximately 80% of the AreA protein in the WT strains. In addition, under the nitrate culture, the content of AreA protein in the nuclei of the WT strains was significantly increased compared with that under the ammonium condition. However, when was silenced, the content of the AreA protein in the nuclei did not change compared with the WT. Compared with the WT, the expression of the glutamine synthetase gene in i-3 and i-48 strains increased by approximately 94% and 88%, respectively, under the ammonium condition, while the expression level of the nitrate reductase gene in i-3 and i-48 strains increased by approximately 100% and 93%, respectively, under the nitrate condition. Finally, silencing inhibited mycelial growth and increased ganoderic acid biosynthesis. Our findings are the first to reveal that a gene from with similarity to the of filamentous ascomycetes contributes to regulating AreA, which provides new insight into how AreA is regulated in .
真菌AreA是氮代谢阻遏（NMR）中关键的氮代谢转录因子。研究表明，在酵母和丝状子囊菌中存在不同的调节AreA活性的方式，但在[具体物种名称未给出]中，AreA是如何被调节的尚不清楚。在此，鉴定出了一个来自[具体物种名称未给出]的与丝状子囊菌的[相关基因名称未给出]具有相似性的基因。根据酵母双杂交试验，NmrA与AreA的C末端相互作用。为了确定NmrA对AreA的影响，使用RNA干扰方法构建了[具体物种名称未给出]的2个沉默菌株，沉默效率分别为76%和78%。沉默[该基因名称未给出]导致AreA含量降低。在铵条件下，i - 3和i - 48中AreA的含量分别比野生型（WT）降低了约68%和60%。在硝酸盐培养条件下，与WT相比，沉默[该基因名称未给出]导致降低了40%。沉默[该基因名称未给出]也降低了AreA蛋白的稳定性。当用环己酰亚胺处理菌丝体6小时时，在[该基因名称未给出]沉默菌株中几乎检测不到AreA蛋白，而在WT菌株中仍有大约80%的AreA蛋白。此外，在硝酸盐培养下，WT菌株细胞核中AreA蛋白的含量与铵条件下相比显著增加。然而，当[该基因名称未给出]被沉默时，与WT相比，细胞核中AreA蛋白的含量没有变化。与WT相比，在铵条件下，i - 3和i - 48菌株中谷氨酰胺合成酶基因的表达分别增加了约94%和88%，而在硝酸盐条件下，i - 3和i - 48菌株中硝酸还原酶基因的表达分别增加了约100%和93%。最后，沉默[该基因名称未给出]抑制了菌丝体生长并增加了灵芝酸的生物合成。我们的研究结果首次揭示了一个来自[具体物种名称未给出]的与丝状子囊菌的[相关基因名称未给出]具有相似性的基因有助于调节AreA，这为[具体物种名称未给出]中AreA的调节方式提供了新的见解。
