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標題: | 建立蠔菇的遺傳及基因體系統以研究蠔菇毒殺線蟲的分子機制 Establishing genetic and genomic resources to study how Pleurotus ostreatus kills nematodes |
作者: | Yi-Yun Lee 李宜芸 |
指導教授: | 薛雁冰(Yen-Ping Hsueh) |
關鍵字: | 蠔菇,正向遺傳篩選,Pleurotus ostreatus, Pleurotus ostreatus,forward genetic screen,toxocyst, |
出版年 : | 2021 |
學位: | 碩士 |
摘要: | Pleurotus ostreatus又稱做蠔菇、平菇或鮑魚菇屬於食線蟲的擔子菌,這類的真菌在低養分的環境下會分泌毒素使線蟲麻痺。在先前的研究中發現秀麗隱桿線蟲 (Caenorhabditis elegans) [1] 碰到P. ostreatus的菌絲時,毒素會藉由線蟲神經細胞上的感覺纖毛進入線蟲體內,造成線蟲頭部急劇收縮及線蟲咽部肌肉和體壁肌肉鈣離子濃度急劇增加,最後導致細胞壞死。然而關於P. ostreatus如何使線蟲麻痺的分子機制依然未知,因此為了要找出與線蟲麻痺相關的基因,我們透過正向遺傳篩選方法,利用ethyl methanesulfonate (EMS) 與UV產生隨機突變,篩選出不會使線蟲麻痺的突變株。我們總共篩選約12,000的突變株,其中有23突變株失去殺死線蟲的能力。利用冷凍掃描式電子顯微鏡 (cryo-SEM) 更進一步的觀察發現大部分的突變株都缺少在野生株P. ostreatus上明顯的球形構造,由此可知此構造在麻痺線蟲的過程中扮演著重要的角色,此構造內有可能存在重要的殺線蟲毒素。我們利用全基因定序及遺傳分析縮小突變基因的範圍並找出可能參與麻痺線蟲的相關候選基因。初步的結果指出平均每一個突變株有107個突變在60個基因上,經過遺傳分析篩選後有146個突變,其中17個基因上的突變較為嚴重,如:失去終止碼、獲得終止碼以及架構轉移突變。其中我們關注於TWF1050突變株上的其中一個基因PC9H_000661含有架構轉移突變,此基因為B型週期素,因此命名為CLB1,我們將此基因剔除,結果發現clb1突變株失去麻痺線蟲的功能,且無法形成球形構造。在未來鑑定毒素的的化學組成是一個重要的方向,我們計畫用此殺線蟲毒素來防治動植物寄生性線蟲。 The oyster mushroom Pleurotus ostreatus is a nematophagous basidiomycete that produces potent toxins to paralyze nematodes when grown in low-nitrogen environments. Our previous study showed that when Caenorhabditis elegans contacted mycelium of P. ostreatus, the toxins could enter through the sensory cilia, causing head muscle hyper-contraction and massive calcium influx in the pharyngeal and body wall muscle, resulting in cellular necrosis. However, the molecular identity of the nematode-paralyzing toxins produced by P. ostreatus remained unclear. To identify the molecular mechanism of P. ostreatus nematocidal activities, we established genetics and genomics analyses and tools to address these questions. First, we conducted random mutagenesis forward genetic screens in P. ostreatus to isolate mutants with defects in paralyzing C. elegans. We generated ~12,000 UV and EMS mutagenized P. ostreatus clones and identified 23 mutants that had lost the nematocidal activities toward C. elegans. Cryo-scanning electron microscopy (cryo-SEM) revealed that most of our mutants did not have the spherical structures that were prominent on the wild-type P. ostreatus hyphae. These results suggest that these structures play important roles in paralyzing nematodes, and likely contain key nematocidal toxins. Whole-genome sequencing and genetic mapping were conducted to narrow down mutation sites that likely contributed to the loss of nematocidal activities. Preliminary analyses revealed that on average, each mutant contained 107 mutations in 60 genes. After filtering by genetic mapping results, we got 146 mutations and 17 of them had severe mutations. These 17 genes would be our priority to conduct the functional study. First, we focused on the TWF1050 mutant, which contained a frameshift deletion in gene PC9H_000661. The gene PC9H_000661 was annotated as a B-type cyclin gene, so we named it CLB1. We further conducted functional analyses of CLB1 and demonstrated that clb1 mutant lost toxicity to nematodes and cannot form the spherical structures, suggesting that CLB1 is required for in the nematocidal activities. In the future, we will first identify the causative mutations in other 22 P. ostreatus mutants by genetic mapping and whole-genome sequencing. Second, we will identify the exact chemical compositions of P. ostreatus toxins, in order to harness the nematocidal potential of P. ostreatus for biological controls of parasitic nematodes. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73871 |
DOI: | 10.6342/NTU202100221 |
全文授權: | 有償授權 |
顯示於系所單位: | 基因體與系統生物學學位學程 |
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