转录因子PaPho与丝状真菌Podospora anserina中磷元素的吸收和利用研究

Phosphorus absorption and utilization in the transcription factor PaPho and the filamentous fungus Podospora anserina

摘要:

作为生物体必需的营养元素之一，磷在物质代谢、信号传导和能量储存中起着关键作用。【目的】研究丝状真菌Podospora anserina中调控磷酸盐代谢相关转录因子的作用，可进一步阐明真核微生物中磷元素吸收的调控机制。【方法】利用同源重组的方法定点敲除P. anserina中2个磷代谢相关转录因子PaPho1和PaPho2，遗传杂交构建双重突变体ΔPaPho1ΔPaPho2；通过表型分析、无机磷含量测定和酸性磷酸酶活性测定分析各突变菌株的变化；利用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）分析磷代谢相关基因的表达情况。【结果】在无机磷作为唯一磷来源的培养基上，ΔPaPho1ΔPaPho2无法生长；在添加有机磷的培养基中，ΔPaPho1ΔPaPho2和野生型菌株生长无显著性差异。在同时添加有机磷和无机磷的培养基中，ΔPaPho1ΔPaPho2的无机磷含量和酸性磷酸酶活性比野生型菌株的分别下降了25.0%和61.9%，ΔPaPho1ΔPaPho2中无机磷酸盐转运蛋白基因的表达水平显著降低。【结论】在P. anserina中，PaPho1和PaPho2作为调控磷酸盐代谢信号通路转录因子，对无机磷的吸收起到关键作用，但并不参与有机磷的代谢调控。该研究可为阐释丝状真菌P. anserina对无机磷吸收的调控机制提供参考。

关键词:丝状真菌;Podospora anserina;转录因子;基因敲除;磷代谢;无机磷

Abstract:

As one of the essential nutrients of living organisms, phosphorus plays a key role in substance metabolism, signal transduction, and energy storage. [Objective] To explore the role of transcription factors related to phosphate metabolism in the filamentous fungus Podospora anserina and further study the regulatory mechanism of phosphorus uptake in eukaryotic microorganisms.[Methods] Two phosphorus-metabolization-related transcription factors PaPho1 and PaPho2 in P. anserina were knocked out by homologous recombination, and a double mutant ΔPaPho1ΔPaPho2 was constructed by genetic hybridization. The changes in mutant strains were analyzed by phenotypic analysis, inorganic phosphorus content determination, and acid phosphatase activity determination. The expression of phosphorous metabolism-related genes was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). [Results] The double mutant ΔPaPho1ΔPaPho2 could not grow in the medium with inorganic phosphate as the only source of phosphorus. There was no significant difference in the growth of ΔPaPho1ΔPaPho2and the wild-type strain in the medium with organic phosphate. In the medium supplemented with organic and inorganic phosphates, the inorganic phosphate content and acid phosphatase activity of ΔPaPho1ΔPaPho2were decreased by 25.0% and 61.9%, respectively, as compared with the wild-type strain. The expression level of inorganic phosphate transporter genes in ΔPaPho1ΔPaPho2 decreased significantly. [Conclusion] In P. anserine, PaPho1 and PaPho2, as transcription factors regulating phosphate metabolism signaling pathway, play a vital role in the absorption of inorganic phosphate, but they do not participate in the metabolic regulation of organic phosphate. This study provides references for the regulatory mechanism of the filamentous fungus P. anserine in the absorption of inorganic phosphate.

Key words:filamentous fungus;Podospora anserine;transcription factors;gene deletion;phosphorus metabolism;inorganic phosphate