應用黑色素基因工程提升蟲生真菌對逆境之耐受能力、致病力及其相關機制

Enhancing the stress tolerance virulence and relevant mechanism of an entomopathogen by metabolic engineering dihydroxynaphthalene (DHN)-melanin biosynthesis genes

曾敏南(Min-Nan Tseng)

指導教授 ： 曾顯雄

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.00004

摘要

蟲生真菌被應用於生物防治工作已有數十年。然而，這些真菌在田間之應用成效並不穩定，其主要原因為環境逆境所造成，這些逆境包括紫外線幅射、極端溫度及乾燥。為解決此瓶頸，本研究由磚格孢菌 (Alternaria alternata) 中選殖出 dihydroxynaphthalene -黑色素 (DHN-melanin) 的生合成基因 (polyketide synthase、scytalone dehydratase及 1,3,8-trihydroxynaphthalene reductase) 並經由農桿菌 (Agrobacterium tumefaciens) 媒介轉殖(Agrobacterium-mediated transformation)，將黑色素生合成基因轉殖進入不具黑色素生合成能力的昆蟲寄生真菌 - 黑殭菌 (Metarhizium anisopliae)。黑色素在黑殭菌轉殖株中的表現，利用分光光度計的方法、液相層析質譜 (LC/MS) 及共軛焦顯微鏡之觀察加以證明。此轉殖株與野生株相比較，對於環境逆境具有較佳的抗性，對於寄主昆蟲亦具有較高的致病力 (virulence)。然而，這些特性的相關機制仍不清楚，因此本研究經由生理及分生的途徑，探討其可能之機制。雖然轉殖株與野生株皆可侵染相同的寄主範圍，但在活體内 (in vivo) 及活體外 (in vitro) 的情況下，轉殖株的分生孢子發芽及產生附著器 (appressorium) 的速率及與產生附著器的數量皆明顯高於野生株。轉殖株對小菜蛾 (Plutella xylostella) 及黃條葉蚤 (Phyllotreta striolata) 的半致死時間 (LT50) 亦顯著的短於野生株。此外，轉殖株對活性氧 (reactive oxygen species, ROS) 的耐受能力較野生株來得高。在in vivo的情況下轉殖株可產生高於野生株40倍的orthosporin，並顯著的大量轉錄chitinase、protease及phospholipase之編碼基因。但轉殖株與野生株相比較之下，附著器的膨壓 (turgor pressure) 及殺蟲毒素destruxin A的產量則有些微下降。轉殖株對黑豆蚜 (Aphis craccivora)、小菜蛾 (Pl. xylostella)、黃條葉蚤 (Ph. striolata) 、銀葉粉蝨 (Bemisia argentifolii) 及東方果實蠅 (Bactrocera dorsalis) 具有高致病力，並具有在植物根部殖據的能力，使轉殖株於田間進行生物防治應用時，應具有較高之潛力。

關鍵字

黑殭菌 ； 黑色素 ； 逆境 ； 紫外線 ； 致病力 ； 小菜蛾 ； 發芽率 ； 活性氧

並列摘要

Entomopathogenic fungi have been used for biocontrol of insect pests for many decades. However, the efficacy of such fungi in field trials is often inconsistent, mainly due to environmental stresses, such as ultraviolet radiation, temperature extremes, and desiccation. To circumvent these hurdles, metabolic engineering of dihydroxynaphthalene (DHN)-melanin biosynthetic genes (polyketide synthase, scytalone dehydratase, and 1,3,8-trihydroxynaphthalene reductase) cloned from Alternaria alternata were transformed into the amelanotic entomopathogenic fungus Metarhizium anisopliae via Agrobacterium-mediated transformation. Melanin express in the transformant of M. anisopliae was verified by spectrophotometric methods, LC/MS and confocal microscopy. In contrast to the wild type strain, the transformant displays a greater resistance to environmental stress and a higher virulence toward target insect host. However, the underlying mechanisms for these characteristics remain unclear; hence experiments were initiated to explore the possible mechanism through physiological and molecular approaches. Although both transformant and wild type strains could infect and share the same insect host range, the former germinated faster and produced more appressoria than the latter, both in vivo and in vitro. The transformant showed a significantly shorter median lethal time (LT50) when infecting the diamondback moth (Plutella xylostella) and the striped flea beetle (Phyllotreta striolata), than the wild type. Additionally, the transformant was more tolerant to reactive oxygen species (ROS), produced 40-fold more orthosporin and notably overexpressed the transcripts of the pathogenicity-relevant hydrolytic enzymes (chitinase, protease, and phospholipase) genes in vivo. In contrast, appressorium turgor pressure and destruxin A content were slightly decreased compared to the wild type. The transformant’s high anti-stress tolerance, its high virulence against five important insect pests (cowpea aphid Aphis craccivora, diamondback moth Pl. xylostella, striped flea beetle Ph. striolata, and silverleaf whitefly Bemisia argentifolii) and its capacity to colonize the root system are key properties for its potential bio-control field application.

並列關鍵字

Metarhizium anisopliae ； melanin ； virulence ； germination rate ； reactive oxygen species ； germination rate ； reactive oxygen species

參考文獻

Chapter 1

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