線蟲捕捉菌之轉錄體分析、基因探勘以及差異性表現探討

Abstract

線蟲捕捉菌可產生各種不同捕捉構造捕捉線蟲，具有發展為生物防治劑防治寄生性線蟲的潛能。然而為了實現此一目的，全面了解線蟲捕捉菌在生態上的適應、生存、寄生以及殖據能力等相關機制極其重要。現代分子生物技術飛躍進展，例如次世代全基因定序技術，提供在線蟲捕捉菌及線蟲寄主在交互作用上，有別於以往且具革命性的新視野。本篇研究中，我們將著重於和捕捉及殖據能力相關基因之探勘，用以闡明解析線蟲捕捉菌之捕捉、侵染、殖據能力，以及共演化歷程。本實驗選取六種代表性線蟲捕捉菌：Arthrobotrys musiformis (黏著網)、 Arthrobotrys brochopaga (收縮環)、Dactylella formosana (黏著球)、 Dactyllela leptospora (黏著球和非收縮環)、Monacrosporium cionopagum (黏著柱)和Monacrosporium phymatopagum (無柄黏著球)；將線蟲捕捉菌作三種處理：不加入線蟲，或加入線蟲培養 24小時或培養48小時。之後抽取核酸，並將六種線蟲捕捉菌不同處理的轉錄體轉成cDNA，利用Illumina/Solexa HiSeqTM 2000定序儀器解序，片段組裝，基因註解和功能分析，以及蛋白質不同資料庫之比對及參與調控代謝途徑比對。將線蟲捕捉菌轉錄體間表現量具有顯著增加或減少的表現差異基因相互比較，篩選出數個具生物學旨趣或應用相關目的之基因進行深度分析。線蟲捕捉菌不同轉錄體間表現量具有顯著差異的基因聯集結果顯示，六種線蟲捕捉菌捕捉構造的形成皆和多醣類、細胞壁外蛋白質、分解合成酵素、過氧小體以及辨識等基因相關。實驗中所得鉅量的生物資訊資料庫，除了使線蟲捕捉菌基礎科學研究更加扎實外，以此所建構之平台可為未來相關研究奠立一個有用的基礎。

Nematode-trapping fungi (NTF) can capture nematodes with varied trapping advices, thus possess potential as biocontrol agent to control parasitic nematodes. Nevertheless, to fulfill this purpose, a thorough understanding of the underlying mechanism to account for the ecological adaption, survival, parasitic and colonizing capability is crucial. In light of the modern rapid advancement of molecular biology and biotechnology, i.e. the next generation whole genome sequencing technique, a completely different revolutionary vision in elucidation of the nematode-trapping fungi interactions becomes feasible. In this study, we focused on de-novo genes discovery in order to correlate to trapping and colonization capability, and evolutionary hierarchy of NTF. We constructed transcriptomes of six representative NTF：Arthrobotrys musiformis (adhesive networks), Arthrobotrys brochopaga (constructing ring), Dactylella formosana (adhesive knobs), Dactyllela leptospora (adhesive knobs and non-constructing ring), Monacrosporium cionopagum (adhesive column), Monacrosporium phymatopagum (sessile adhesive knobs) without or with nematode amendment and incubation for 24, 48 hrs, respectively. The constructed cDNA libraries were read by Illumina/Solexa HiSeqTM 2000 sequencing system, then sequences assembled, annotated and function analyzed. The significantly up-regulated and down-regulated genes were subject to comparison between or among transcriptomes. Several genes suspected to comply with the purpose mentioned above were acessed and proceeded for in-depth analysis, including protein database blasting, prediction, metabolic pathway assessment and putative functional analysis. Comparative transcriptomes analysis revealed that some genes were significantly up-regulated in all NTF, such as phosphorylase, cell surface glycoprotein, large adhesion, peroxisome and molecular involved in recognition. The immense bioinformatics came up from this study actually established a useful platform for perspective study.