| 摘要: | 許多植物病原真菌會產生毒素而使病原菌可成功的寄生於寄主植物上， Alternaria、Cercospora、Cladosporium、Elsinoe，及Hypocrella 等重要病原真菌會產生 perylenequinone 類的毒素，Cercosporin(尾孢菌素)是此類毒素中被研究較多的模式系統，已有實驗證實cercosporin 在病原性上扮演極重要的角色。此毒素在光照的情況下會使氧氣產生單重態氧(singlet oxygen, 1O2)及超氧自由基(superoxide O2 -)，因而造成植物細胞膜的脂質過氧化、細胞膜滲漏及細胞死亡。cercosporin 生合成受光照、營養源、溫度、環境酸鹼值、金屬離子等的調控。在分子層次上目前已知由八個基因(cercosporin toxin biosynthesis, CTB)組成的基因簇(gene cluster)為合成cercosporin 所須；CRG1 及 CTB8 為轉錄調控子，調控cercosporin 的產生； MAP kinase 訊息途徑與cercosporin 合成有關。此外，cercosporin 為紅色的毒素，若破壞合成cercosporin 所需的CTB 基因，突變菌株呈現不同顏色。因此，可利用CTB 基因的突變系統，探討不同因子對破壞特定基因(CTB)的效率。本計畫為期二年，主要目的有三，第一是以C. nicotianae 的CTB 基因為目標基因，探討基因轉殖時所用的不同disruption construct(含circular plasmid、 linear plasmid、whole PCR product、split marker、linear minimal element 如圖一所示) 及其他因子對目標基因之破壞效率；其次是要進一步探討不同氮素源、碳素源、金屬離子、胺基酸、酸鹼值等對cercosporin 生合成之影響，以及在分子層次上是否透過 CTB8、CRG1、MAP kinase 等基因調控。此外，本實驗的另一個目的是要進一步選殖 C. nicotianae 之calmodulin 基因，利用基因突變的方式，研究calmodulin 基因如何調控cercosporin 的合成，以及calmodulin 基因與CTB8、CRG1、MAP kinase 等調控基因的相互關係。本計畫在基因轉殖的成果，可加速Cercospora spp.及其他真菌之基因轉殖技術的開發。在cercosporin 的生合成之研究成果，因為cercosporin 是 perylenequinone 類毒素的指標系統，若對cercosporin 有更詳盡的研究，未來必有利於此類病害的防治工作。
Many plant pathogenic fungi produced toxins for their successful pathogenesis. Light-activated perylenequinone toxins are produced by a number of important fungal plant pathogen, including species of Alternaria, Cercosporin, Cladosporium, Elsinoe, and Hypocrella. Cercosporin is the best-studied perylenequinone toxin and has been demonstrated to play important role in Cercospora disease. Under light condition, the toxin absorbs light energy and reacts with oxygen to produce activated oxygen species such as singlet oxygen (1O2) and superoxide (O2 -). The activated oxygen causes lipid peroxidation of cell membranes, electrolyte leakage, and eventually cell death. Production of cercosporin is affected by light, nutrient conditions, temperature, pH of environment, and ions. Recently studies indicated that CTB (cercosporin toxin biosynthesis) gene cluster, comprised of eight genes required for cercosporin biosynthesis. In addition, biosynthesis of cercosporin is regulated by transcriptional factors, CRG1 and CTB8, and MAP kinase. In addition to study of cercosporin biosynthesis, Cercospora nicotianae is a good system to investigate and improve fungal transformation. Disruption of CTB genes result in loss of cerosporin synthesis and the CTB mutants are easily identified by color of fungal colony. There are three major purposes in this 2-year project. First of all, CTB gene cluster will be used as visible markers to investigate the effect of disruption constructs and other factors on gene-disruption efficiency. The disruption constructs of circular plasmid, linearlized plasmid, whole PCR fragment, and split maker will be generated in this project. Second, the production of cercosporin regulated by factors such as nitrogen sources, carbon sources, metal ion, amino acids, and pH value will be characterized.We will further analyzed if such regulation is controlled by CRG1, CTB8, or MAP kinase. Thirdly, calmodulin gene of C. nicotianae will be cloned and disruption construct will be generated to investigate the role of colmodulin in cercosporin synthesis. The information of fungal transformation of C. nicotianae can be applied to other fungi and enhance the efficiency of gene characterization. Besides, a greater knowledge of controlling cercosporin synthesis may lead to better management for perylenequinone-related fungal disease. |
