應用黑色素生合成基因研發鑑定炭疽病菌之生物晶片及 探討其類緣關係

Using dihydroxynaphthalene melanin biosynthesis genes in developing biochip for identification and phylogenetic study of Colletotrichum

胡賢彬(Hsien-Pin Hu)

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/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01045

摘要

傳統上炭疽病菌屬(Colletotrichum)之鑑定主要依據植物寄主範圍及型態構造特徵，但常因重疊導致分類混淆和鑑定困難，甚至利用單一基因當為分子標記，用以診斷鑑定不同種的炭疽病菌，但因序列彼此相似度太高、解序力低而無法區別並釐清親緣關係。目前多基因座分析為探討炭疽病菌類緣及種間鑑定之主要方法，但需耗費較多人力、物力與時間。為克服此種障礙，遂進行探討與致病性相關之黑色素生合成基因，在炭疽病菌診斷鑑定及類緣關係應用之可能性。研究結果顯示炭疽病菌屬之黑色素生合成基因Scytalone dehydratase(SCD)在種間相似度為70~100%、1,3,8-trihydroxynaphthalene reductase(THR)則為78~100%，相似度不高有利於類緣分析及種間鑑定。此外，兩基因皆具有較長片段的內插子，也有利於設計寡核苷酸探針。分別應用SCD(651 bp)及THR(1,286 bp)基因片段，以RaxML之最大似然性分析，建構59種代表性炭疽病菌，皆可將其區分為9個支序群(clade)，且類緣樹末端種間分支少有多支性，顯示此二基因皆可用以區辨不同種之炭疽病菌。利用SCD進一步探討感染草莓、紅棗之51株炭疽病菌分離株，47株屬於gloeosporioides clade，4株則屬boninense clade，其病原皆非以往所記載之C. gloeosporioides、C. acutatum、C. theobromicola (syn. C. fragariae)，其中除C. asianum/C. fructicola、C. boninense外，位在gloeosporioides clade中出現頻率89%之菌株可能為一新種。另外ML443、ML463、ML348對草莓之病原性也經柯霍氏法則(Koch's postulates)證明。此分離株可以感染草莓，並造成草莓冠部褐化、全株萎凋。此外，利用黑色素生合成基因SCD，所完成設計之寡核苷酸生物晶片，可鑑別28種涵蓋國內外嚴重危害具有經濟價值農園藝作物之重要炭疽病菌。也應用SCD基因當為炭疽病菌分子標記，試圖解決由食品工業研究所之生物資源保存及研究中心所保存之26株炭疽病菌身分及分類問題，並提出應修訂的菌株及可能為特有種之名單。未來將進一步蒐集文獻序列，分析更多炭疽病菌菌株，應用黑色素生合成基因或多基因座分析釐清此26株炭疽病菌株之屬性。本研究結果顯示黑色素生合成基因可資應用於炭疽病菌屬之類緣分析，而所完成設計之生物晶片，未來將有助於炭疽病菌之防檢疫、生態學、病害防治管理及流行病學等研究。

關鍵字

炭疽病菌 ； 黑色素生合成基因 ； 生物晶片

並列摘要

Traditionally, identification of the Colletotrichum is mainly based on the morphological characters and host specificity, but often confused due to the overlap of identification parameters. The same status also occurs to the ITS molecular markers, owing to the high similarity of gene sequence, resulting in the low resolution in taxa discrimination and phylogenetic analysis. Even though more recently the use of multilocus genes as a barcode for the study of Colletotrichum are widely accepted, however, their usage are limited by its high expenditure of resources. To circumvent these obstacles, attempt using the pathogenesis correlated dihydroxynaphthalene (DHN)-melanin in clarification of the taxonomy and phylogeny of Colletotrichum were initiated. The outcome revealed the similarity of DHN-melanin biosynthesis genes scytalone dehydratase (SCD) and 1,3,8-trihydroxynaphthalene reductase (THR) among Colletotrichum range from 70-100% and 78-100%, respectively. The low sequence similarity will facilitate taxonomic and phylogenetic study of Colletotrichum. In other aspects, the long length of introns of these genes also favor the relvant study. By using the SCD (651 bp) and THR (1,286 bp), the 59 representative Colletotrichum species can be unambiguously divided into nine clades without terminal polytomy in constructed tree inferred by the RaxML method. The results implicated the great potential of SCD and THR genes in fabricating biochip. Furthermore, investigation of the 51 Colletotrichum strains isolated from infested strawberry and red jujube seedlings using SCD as barcode, which clearly indicated the causal organisms were not C. gloeosporioides, C. acutatum, C. theobromicola (syn. C. fragariae), instead, except C. asianum/C. fructicola and C. boninense, one of the endemic strain located in gloeosporioides clade with 92% incidence might be the causal pathogen. Besides, the pathogenicity of the Colletotrichum ML348, ML443, ML463 towards strawberry was demonstrated via Koch’s postulates. These strains could infect strawberry and led to the root crown browning and seedling wilting. Additionally, by using DHN-melanin SCD gene, a biochip developed could simultaneously, rapidly and precisely identified 28 crucial Colletotrichum species, which could devastate the crucial agronomic and horticultural crops worldwide. Likewise, by SCD bacode, 26 endemic Colletotrichum species deposited in Bioresource Collection and Research Centre, FIRDI, their identity were clarified. To justify our sanction, additional references, sequence, and Colletotrichum holotype or epitype species will be collected, sequenced and analysed. In conclusion, the DHN- melanin biosynthesis genes can be used in phylogenetic study of Colletotrichum, and the biochip developed can be used in quarantine, ecology, disease control and management, and epidemiology study.

並列關鍵字

Colletotrichum ； DHN-melanin biosynthesis gene ； Sytalone dehydratase ； 1,3,8-trihydroxynaphthalene reductase ； oligocleotide microarray.

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