透過正向遺傳篩選選出對線蟲捕食缺陷之線蟲捕捉菌

Abstract

線蟲捕捉菌在演化的過程中面對養分缺乏的環境時，選擇了線蟲作為捕食的對象。他們藉由長出複雜的捕蟲構造抓住且殺掉線蟲，並將這些線蟲作為額外的碳與氮的養分來源。我們實驗室以Arthrobotrys oligospora與秀麗隱桿線蟲(Caenorhabditis elegans)為研究對象，分別做為獵食者與被獵食者，探討兩者之間的交互作用關係。為了更進一步地瞭解線蟲捕捉菌如何感知環境中的線蟲與其捕蟲網的生成機制，我們藉由正向遺傳學的篩選(Forward genetic screen)找出無法形成捕捉構造的Arthrobotrys oligospora突變株。利用ethyl methanesulfonate與UV作為致突變劑，我們總共產生了5552個突變株，並在最後挑出16個在捕捉線蟲上具有明顯缺陷的突變株。接著我們對這16個突變株做全基因體定序(whole-genome sequencing) 找到所有突變位置，並以此看哪些突變的基因使他們失去捕捉秀麗隱桿線蟲的能力。經過篩選分析顯示這些突變多與訊息傳遞、轉錄、或膜上運輸有關。我們針對選出的目標基因做基因剃除來檢視這些基因是否參與線蟲捕捉菌捕捉線蟲的過程。目前，我們發現一個具PH domain的基因與線蟲捕捉菌捕捉網的生成有關。預期在這些篩選出的目標基因中，我們除了會找到已知在其它致病性真菌中扮演重要角色的基因，也會發現線蟲捕捉菌自身特有的基因。

Nematode-trapping fungi are a group of nematophagous fungi that have evolved to prey on nematodes by developing complex trap structures under nutrient-poor environments; the traps can capture and kill the nematodes, providing additional nitrogen and carbons sources. We used Arthrobotrys oligospora and Caenorhabditis elegans as models to study the interactions between preys and predators. To identify genes and pathways that are required for sensing the nematodes and trap morphogenesis, we conducted a forward genetic screen in A. oligospora to isolate mutants with defects in trapping C. elegans. We generated 5552 EMS and UV mutants and identified 16 mutants with strong defects in trap morphogenesis. Whole genome sequencing and bioinformatic analyses for the 16 mutants identified the potential mutations that affect trapping ability. Preliminary data demonstrated that mutations in genes that have a role in signaling, transcription or membrane transport might affect trap morphogenesis. Targeted gene deletion mutants were generated to investigate the roles of these candidate genes during nematode-trapping process. So far, we found one unstudied gene predicted to contain a PH domain to influence trap morphogenesis. In addition to genes that conserved in other pathogenic fungi, we have also identified novel genes that are unique in nematode-trapping fungi might play important roles in their predatory behaviors.