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標題: | 銨轉運蛋白於寡孢節叢真菌之功能探討 The Roles of Ammonium Transporters in the Nematode-Trapping Fungus Arthrobotrys oligospora |
作者: | 阮聖茜 Sheng-Chian Juan |
指導教授: | 薛雁冰 Yen-Ping Hsueh |
關鍵字: | 銨轉運蛋白,寡孢節叢真菌,Arthrobotrys oligospora,線蟲捕捉菌, Ammonium transporter,Arthrobotrys oligospora,Nematode-trapping fungi, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | 線蟲捕捉菌為一群在養份缺乏的環境下特化出捕捉構造來捕捉線蟲為食的真菌,其中,在台灣廣泛分佈的寡孢節叢真菌Arthrobotrys oligospora以立體的黏著網來捕捉線蟲,也逐漸成為研究食性交互作用的模型之一。銨離子是微生物主要的氮元素來源之一,然而銨也會抑制A. oligospora的黏著網形成。銨轉運蛋白家族(Amt/Mep/Rh family)在各個物種之間相當保守,擁有相似的蛋白質序列及結構。為了瞭解銨是如何運輸以及影響黏著網的形成,我們在A. oligospora找到三個銨轉運蛋白並依據序列相似度分別命名為MEP1、MEP2以及MEP,並推測MEP2與MEP3有較高的銨親和性,而MEP1則為銨低親和性的轉運蛋白。在time-course RNA-seq的結果中,MEP基因在A. oligospora捕食線蟲的前期提高表現量,於後期降低表現量,此外,MEP基因也受到銨離子的抑制,表示MEP基因可能具有感知環境及運輸銨離子的功能。為了暸解其功能,我們將MEP基因剔除並發現突變株對於線蟲的敏感性降低而形成較少的黏著網;相反的,相較於野生型,突變株在添加銨離子的環境下依然能形成黏著網。而突變株在形態與線蟲捕捉功能上則與野生型無異。當將A. oligospora的MEP基因表現於Saccharomyces cerevisiae的mep2與mep1mep2mep3突變株,可以分別在低銨離子濃度環境下恢復假菌絲的形成與菌落生長。以上實驗結果表示A. oligospora的Mep銨轉運蛋白在捕捉線蟲的過程中扮演感知以及運送銨離子的功能。此外,我們發現mep1突變株的產孢過程有延遲,推測MEP1基因除了捕捉網的形成以外對於產孢過程也具有正向的影響。 Nematode-trapping fungi develop various devices to trap and consume nematodes as a food source under nutrient-limiting conditions. Arthrobotrys oligospora is the most abundant species in Taiwan that produces adhesive networks to catch nematodes and it has become a model organism for investigating predator-prey interactions. Ammonium is a major nitrogen source for microbes, and it suppresses trap formation by A. oligospora. The Ammonium transporter (Amt)/Methylammonium permease (Mep)/Rhesus protein (Rh) family is conserved across all organisms, sharing similar sequences and structures. To investigate how ammonium is transported and affects trap formation by A. oligospora, we identified three potential ammonium transporters in this fungus, i.e., MEP1, MEP2, and MEP3. Phylogenetic analysis revealed that MEP2 and MEP3 are both high-affinity transporters, whereas MEP1 is a low-affinity transporter. Based on time-course RNA-sequencing data, the three MEP genes are upregulated when nematodes are sensed, and they are downregulated upon prey capture, indicating that they may play a role in sensing prey presence. Additionally, their expression is suppressed by nitrogen treatment, unlike the case for carbon application, supporting the importance of intracellular nitrogen levels in sensing starvation. To investigate their functions, we deleted the MEP genes individually and found that trap formation in response to nematodes was impaired in the mep mutants, though trap morphology and adhesiveness were not affected. However, ammonium treatment rescued trap formation by the mep1 and mep1mep2 mutants. Heterologous gene expression in Saccharomyces cerevisiae mep2 and mep1mep2mep3 mutants complemented pseudohyphal and growth defects under low ammonium conditions, respectively. Additionally, absence of mep1 resulted in delayed conidiation. Together, these results suggest that the Mep transporters are involved in ammonium sensing and transportation in A. oligospora, with Mep1 potentially playing a positive role in the conidiation process. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88359 |
DOI: | 10.6342/NTU202301993 |
全文授權: | 同意授權(限校園內公開) |
顯示於系所單位: | 基因體與系統生物學學位學程 |
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