蝴蝶蘭PaCOI1基因之選殖及參與茉莉酸訊息傳導及抗病性研究

Abstract

蝴蝶蘭屬（Phalaenopsis）蘭科植物為台灣外銷產值最高的花卉作物，但其於栽培過程中可能受到病原菌之感染危害，造成生產者的重大損失。Coronatine-insensitive 1 gene (COI1)為jasmonic acid (JA)路徑重要的基因，主要功能為感受細胞中JA濃度，並調控JA下游基因的表現，如抗病性基因、老化基因、根部發育、雄性花器成熟等。本實驗利用阿拉伯芥COI1基因核酸序列，進行蝴蝶蘭Expressed sequence tag (EST) library之比對，得到256 bp長度之序列片段，進一步利用Rapid Amplification of Complementary DNA Ends (RACE) 方法，得到蝴蝶蘭PaCOI1基因之cDNA全長，預測轉譯出585個胺基酸之蛋白質。親緣關係比對結果顯示，蝴蝶蘭PaCOI1 胺基酸序列與已發表的其他物種中，較親近於同為單子葉植物的小麥及水稻同源基因。在基因表現方面，蝴蝶蘭PaCOI1基因於根、莖、葉、花梗、花等部位，可偵測到其表現；在傷口處理與MeJA施用的情形下，PaCOI1所調控之PaJAZ1與PaMYC2基因可於2小時內，快速提高其表現量，而因傷口所引發PaJAZ1 和PaMYC2 大量mRNA累積之現象，與PaCOI1基因相關。而因應不同病原菌的感染，PaJAZ1與PaMYC2基因表現上，亦有所差異。為了解PaCOI1基因之功能，分別建構靜默(silencing)與過度表現(overexpression)載體，並應用Agrobacterium-mediated transient gene expression and silencing工具，進行蝴蝶蘭中PaCOI1基因表現調控影響之觀察。VIGS 靜默植株PaCOI1表現量約可降低至3到4成，接種黃葉病菌於PaCOI1基因靜默植株，呈現感病加劇之情形。黃葉病菌與軟腐病菌於PaCOI1過度表現植株中，病斑發展並無顯著差異。利用 Fast Agro-mediated Seedling Transformation (FAST) 方法，已成功於阿拉伯芥中表現PaCOI1基因。本研究結果證實，蝴蝶蘭PaCOI1基因為病原菌感染及傷口所引起JA反應路徑之重要基因。

Phalaenopsis belongs to Orchidaceae, and is one of the most valuable flower crops for sale abroad in Taiwan. In orchid nursery, Phalaenopsis is subjected to infection by many pathogens during different growing periods, and severe economic losses to growers often occur. Coronatine-insensitive 1 gene (COI1) is responsible of sensing jasmonic acid (JA) inside cells, and regulates downstream JA-responsive genes involved in disease resistance, senescence, root development, and male fertility. In this study, we utilized Arabidopsis COI1 gene sequence to search against Phalaenopsis EST library, and identified a partial 256 bp fragment of Phalaenopsis COI1 homologue. By Rapid Amplification of Complementary DNA Ends (RACE) methods, the full length of Phalaenopsis aphrodite COI1 (PaCOI1) cDNA was obtained and found PaCOI1 encodes a 585 amino acid residues protein. Phylogenetic analysis revealed that PaCOI1 is more closely related to homologues of monocot plants such as Oryza sativa and Triticum aestivum. The basal PaCOI1 transcript levels can be detected in root, stem, leaf, stalk and flower. After wounding and exogenous MeJA treatments, PaJAZ1 and PaMYC2 genes, two downstream targets in JA signaling pathway, were quickly and dramatically induced within two hours. Wound-induced accumulation of e PaJAZ1 and PaMYC2 mRNAs was largely dependent on PaCOI1. In response to pathogen infection, PaJAZ1 and PaMYC2 were also differentially regulated. In order to determine the function of PaCOI1 gene, silencing and overexpression vectors were constructed and Agrobacterium-mediated transient gene expression and silencing systems were used. Gene expression studies confirmed that PaCOI1 in VIGS silencing plants was reduced to 30-40％ of the wild-type level. When challenging silencing plants with different pathogens, more severe symptoms were found by F. solani infection. No significant difference in lesion development of PaCOI1 overexpression plants was found when infected with F. solani or E. chrysanthemi. Finally, Fast Agro-mediated Seedling Transformation (FAST) method was successfully employed to deliver and express PaCOI1 in Arabidopsis. Our studies demonstrate that PaCOI1 is an important P. aphrodite gene involved in pathogen and wounding induced JA signaling pathway.