探討線蟲捕捉菌絲氨酸蛋白酶基因之特性及開發林木褐根病菌檢測用生物晶片

Abstract

真菌廣泛存在於地球上，扮演多重角色，可為人類的朋友或敵人。真菌不僅是生物系統最主要的分解者，它們亦是食品、藥物以及發酵工業的重要微生物資源，更是探索、闡明和理解生物學的模式生物之一。反之、真菌不僅會引起食物腐壞，也是人類、動物及植物致病因子。此論文涵蓋真菌基礎及應用之研究、探討食性演化的分子生物學模式、生物防治應用之相關基因篩選、以及森林真菌病原之檢測。其內容主要在探討於線蟲捕捉菌 Arthrobotrys musiformis 之絲氨酸蛋白酶基因AmSp1 的選殖、特性界定及親緣分析；線蟲捕捉菌 Arthrobotrys javanica 絲氨酸蛋白酶基因 AjSP1 的選殖和特性界定，及其於形成肉食性行為演化可能扮演之角色；以及應用隸屬於擔子菌門 (Basidiomycota) 之木層孔菌(Phellinus) 之核苷酸 ribosomal ITS 基因，建構快速診斷鑑定晶片平台。 此研究顯示絲氨酸蛋白酶於線蟲捕捉菌之粘性捕捉網之形成、分化、捕捉以及殖據和存活能力所扮演之多重角色；而所建構之木層孔菌快速診斷鑑定晶片平台，應有助於林木褐根病菌 Phellinus noxius、層根腐病菌 Phellinus weirii 等重要病原之早期診斷鑑定、生態學和防治等之應用。

Fungi are ubiquitous in this world serving both as friend and foe. Not only are fungi ultimate decomposers giving regeneration to life’s matters, they are a source of food and medicine, act in many of the fermentation and biochemical processes essential to our everyday life, and serve as fundamental model organisms in elucidating our basic understanding of biology and science. On the other hand, fungi can be equally detrimental on the opposing end in causing spoilage to food, and diseases to both humans, animals and plants. The scope of this thesis centers on researches in which fungi were utilized as a model for understanding the fundamental biology in feeding trait development, molecular characterization and cloning of genes in potentially serving a role for biocontrol applications, as well as research pertaining to the development of robust platforms to detect fungal pathogen. In this thesis, we report on key researches entailing the cloning and characterization of cuticle-degrading serine protease gene, AmSp1, in nematode-trapping fungus Arthrobotrys musiformis; the cloning and characterization of cuticle-degrading serine protease gene, AjSp1, in the development of carnivorous feeding trait in nematode-trapping fungus, A. javanica; and the development of oligonucleotide microarray platform for the simultaneous and specific detection of seventeen important Phellinus species. The result of our findings first implicate that the identified serine protease gene, AmSP1, had undergone purifying selection and contained a high degree of conservation of the catalytic and binding sites with previously described serine proteases from other nematode-trapping fungi. Furthermore, AjSP1, the homolog of AmSP1 in A. javanica was found to be pivotal in the development of adhesive net traps and nematode-capturing capacity of A. javanica. Last but not least, the developed Phellinus microarray system was highly sensitive, robust, and efficient, as confirmed against template DNA from two key Phellinus species, as well as field samples collected from tree roots and trunks. The results of our findings may serve as an incremental step in the endeavor of mycological research and science.