Transcriptional regulation of the antifungal protein in Aspergillus giganteus
Transcriptional regulation of the antifungal protein in Aspergillus giganteus
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DOI：
10.1007/s00438-001-0609-6
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发表时间：
2002-01-01
影响因子：
3.1
通讯作者：
Stahl, U
中科院分区：
文献类型：
--
作者：
Meyer, V;Wedde, M;Stahl, U
使用报告系统研究了编码巨曲霉抗真菌蛋白的 afp 基因的表达调节。为此，将编码 β-葡萄糖醛酸酶 (GUS) 的大肠杆菌报告基因 uidA 置于 afp 启动子的控制之下。在测试的 156 个转化体中没有观察到报告构建体与 afp 位点的同源整合。在携带单个异位整合构建体副本的转化体之一中，GUS 和 AFP 在各种培养条件下都显示出完全相同的时间表达模式，如 Northern 和蛋白质分析所测定。因此，该转化体用于鉴定参与 afp 表达转录调节的因子。仅在营养菌丝体中可检测到表达，而气生菌丝或分生孢子中没有表达，表明 afp 表达受到发育调节。 afp 的转录受环境 pH 值调节，在酸性条件下受到抑制，在碱性条件下强烈诱导。这一观察结果表明 PacC 调节 afp 基因，这与 5' 上游区域内两个推定的 PacC 结合位点的存在一致。转录不受碳分解代谢物抑制或氮代谢物抑制。在过量氯化钠和乙醇存在下以及碳饥饿条件下生长时，热休克可上调 afp 的表达。相反，在过氧化氢存在和氮饥饿条件下表达略有下降。这些数据与 afp 启动子内假定的热休克元件 (NTTCNNGANTTCN) 和五个假定的 C4T 应激反应元件的存在相一致。
Regulation of the expression of the afp gene that codes for the Antifungal Protein of Aspergillus giganteus was investigated using a reporter system. For this purpose, the E. coli reporter gene uidA encoding beta-glucuronidase (GUS) was placed under the control of the afp promoter. No homologous integration of the reporter construct into the afp site was observed among 156 transformants tested. In one of the transformants carrying a single, ectopically integrated, copy of the construct, GUS and AFP both displayed exactly the same temporal expression patterns under various cultivation conditions, as assayed by Northern and protein analyses. Thus, this transformant was used to identify factors that are involved in the transcriptional regulation of afp expression. Expression is only detectable in the vegetative mycelium, whereas no expression occurs in aerial hyphae or conidia, indicating that afp expression is developmentally regulated. Transcription of afp is regulated by ambient pH, being suppressed under acidic conditions and strongly induced under alkaline conditions. This observation suggests that PacC regulates the afp gene, which is consistent with the presence of two putative PacC binding sites within the 5' upstream region. Transcription is not subject to carbon catabolite repression or nitrogen metabolite repression. The expression of afp is up-regulated by heat shock, upon growth in the presence of excess NaCl and ethanol, and under conditions of carbon starvation. In contrast, expression decreases slightly in the presence of hydrogen peroxide and under nitrogen starvation. These data are compatible with the presence of a putative heat shock element (NTTCNNGANTTCN) and five putative C4T stress-responsive elements within the afp promoter.