| 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，此毒素在病原性上扮演極重要的角色。由C. nicotianae 所產生的毒素cercosporin，是此類毒素中被研究較多的指標模式系統，已有實驗證實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，可能與 calmodulin 訊息途徑有關。在分子層次上，目前已知由八個基因(cercosporin toxin biosynthesis, CTB)所組成的基因簇(gene cluster)參與cercosporin 之生合成；然而，在 cercosporin 生合成的調控上並無太多研究，目前只知CTB8 為轉錄調控子，調控其他 CTB 基因的表現及cercosporin 的產生； MAP kinase 訊息途徑與cercosporin 合成有關。因此，本研究擬以C. nicotianae 為模式，深入研究cercosporin 生合成的調控。本計畫為期三年，第一年是要探討不同氮素源、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 kinase kinase(MAPKKK)等基因調控，並完成calmodulin 基因突變菌株之篩選，探討calmodulin 基因在cercosporin 合成所扮演之角色。第二年的目標則要進一步探討其他未知基因在 cercosporin 生合成調控所扮演之角色，將C. nicotianae 分別於光照及黑暗培養，利用抑制性扣減雜交法(suppressive subtractive hybridization)，建立會在光照下大量表現的基因庫，所獲得的基因就有可能與cercosporin 生合成調控有關，並進一步進行DNA 解序、利用生物資訊學的方法進行基因功能的預測，選殖可能與cercosporin 生合成調控有關之基因。第三年除了繼續進行相關新調控基因之選殖外，欲完成這些新基因之突變構築 (disruption construct) 及突變菌株之篩選，確認這些新調控基因對cercosporin 產量的影響，並進一步測量各突變菌株之CTB8、CTB gene cluster、CRG1、MAPKKK、 calmodulin 等已知基因的表現，以研究各個新調控基因調控cercosporin 生合成的路徑；此外，可利用C. nicotianae 特定基因(如CTB1、CRG1、calmodulin、及新調控基因等) 之突變菌株，測量突變菌株之其他基因表現是否被影響，如此可釐清各個調控基因之上下游關係，相信未來必可構畫出cercosporin 生合成調控的藍圖，以及建構 perylenequinone 類毒素生合成調控之指標系統。經由本研究，我們將對cercosporin 生合成調控有更詳盡的研究，相信未來對於perylenequinone - producing 的病原之病害防治工作必有重大的貢獻。
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. However, not many studies focus on the regulation of cercosporin production. Biosynthesis of cercosporin is regulated by transcriptional factors, CRG1 and CTB8, and MAP kinase kinase kinase (MAPKKK). The objective of this 3-year project is to investigate the regulation of cercosporin production in Cercospora nicotianae. In the first year progress, 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 MAPKKK. In addition, calmodulin gene of C. nicotianae will be cloned and disruption construct of calmodulin gene will be generated. The fungal mutants with defective calmodulin gene will be created to investigate the role of colmodulin in cercosporin synthesis. In the second year progress, the novel genes involved in control of cercosporin production will be identified. C. nicotianae produces cercosporin under light condition, but no cercosporin production when C. nicotianae is cultured under dark condition. The cDNA highly expressed under light condition will be cloned by suppressive subtractive hybridization. BLAST algorithms will be used to identify protein homologous sequence using GenBank’s nonredundant databases, and the COGEME database (Consortium of Functional Genomics of Microbial Eukaryotes). In the third year progress, we tend to clone the full length of novel genes and characterize the function of these novel genes in regulation of cercosporin production. The gene expression of CTB8, CTB gene cluster, CRG1, MAPKKK controlled by novel genes will be investigated. In addition, the regulation network of cercosporin production, controlled by CTB1, CRG1, MAPKKK, and newly identified regulatory gene, will be investigated by studying the individual gene expression in different fungal mutants. We strongly believe that the regulation of cercosporin biosynthesis will be clearer through this project. Besides, a greater knowledge of controlling cercosporin synthesis may lead to better management for perylenequinone-related fungal disease. |
