摘 要

　　在本實驗室的先期相關研究中，已選殖出兩個逆境訊息傳遞相關基因，包括PaMAPK1及PaMEK1，並且發現其與低溫及組織特異性有關。在本篇研究中，我們從蝴蝶蘭中選殖出三個逆境訊息傳遞相關基因，包括PaSIPK、PaCDPK1及PaPTP1，以及PaMAPK1與PaCDPK1的啟動子部位。PaSIPK、PaCDPK1及PaPTP1的親源演化分析結果指出，PaSIPK屬於Group A2之MAPK，PaCDPK1屬於Group I之CDPK，PaPTP1則可能屬於Class I Cysteine-Based PTPs；基因分析結果顯示，其等在蝴蝶蘭的基因組中是單一拷貝基因，且PaCDPK1及PaPTP1分別在花部的唇瓣及蕊柱有組織特異性。軟腐病的處理會誘導PaMAPK1及PaCDPK1表現增加，但PaMEK1及PaPTP1則有減少的情況。此外，PaCDPK1亦會受低溫誘導表現。由以上結果推測，PaMAPK1、PaMEK1、PaCDPK1及PaPTP1在蝴蝶蘭發育過程、花部特化及遭受逆境之情形下，均可能扮演著調控的角色。此外，PaCDPK1靠近感染部位有高量表現，與一般狀況相差甚多，可見其啟動子對於病原菌有相當大量的反應。在本篇研究中所選殖出之PaPTP1，是目前已知第一個單子葉植物的PTP，其重組蛋白能快速水解pNPP(pyronitrophenyl phosphatase)，顯示具有TP之活性。本篇論文不僅選殖出蝴蝶蘭中訊息傳遞途徑相關的部分基因並初步分析之，更期望所得的結果能對相關產業有所貢獻。

Abstract

　　Plants are constantly exposed to a variety of biotic and abiotic stresses. To survive under these challenges, plants have developed elaborate signaling transduction mechanisms to perceive external signals and manifest adaptive responses with proper physiological and morphological changes. The mitogen-activated protein (MAP) kinase cascade, calcium-dependent protein kinases (CDPKs) and protein tyrosine phosphatases（PTPs）are important intracellular signaling modules. The interactions between pathogen and host-plant are very complex because of the ability to recognize each other and varied resistant mechanism. The industry of Phalaenopsis in Taiwan is internationally competitive and creates lots of benefit in foreign-trade market every year. However, the serious pathological problems, especially caused by the Erwinia spp., result in enormous disadvantage. Therefore, we are trying to figure out the interactions between Erwinia spp. and orchids, and the performance underlying signal transduction pathway of plants attacked by pathogens.
Two stress-related genes－PaMAPK1 and PaMEK1 had been isolated by our labatory from the Phalaenopsis amabilis. Their expressions were regulated under the cold stress and tissue specific. In this research, we have isolated another three sress-related genes－ PaSIPK、PaCDPK1 and PaPTP1 from the P. amabilis. The corresponding promoters of PaMAPK1 and PaCDPK1are also identified. According to the phylogenetic analysis, PaSIPK and PaCDPK1 both involved in the stress defense mechanism. Southern blotting analysis suggested that they were single-copy genes in orchid. Northern blot analysis showed that they were predominantly expressed in flower of orchid. The transcript levels of PaMAPK1 and PaCDPK1 increased markedly while PaMEK1 and PaPTP1 decreased slightly when plants were treated with Erwinia. Besides, PaCDPK1 were also rapidly induced by 4℃ treatment. These results suggest that they may function both in the developmental stages and in stress-responsive pathway of orchids. The transcript levels of PaCDPK1 were quite low in all tissues but highly expressed in the water-soaked infected region, showing that its promoter may very sensitive to pathogen. The PaPTP1 cloned from orchids in this research was the first PTP isolated in monocots, and the recombinant PaPTP1 hydrolyzed pNPP (pyronitrophenyl phosphatase) quickly, showing that PaPTP1 is a highly active phosphatase. Genetic engineering of signal transduction mechanisms is starting to emerge. This approach will not only contribute to the understanding of signaling systems in plants but will eventually contribute significantly to agricultural practice.

林婉嬪 (2003) 水稻及蝴蝶蘭中MAPK訊息傳遞基因之選殖及特性分析。國立成功大學生物學研究所碩士論文。

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