乙烯(C2H4)是一重要的植物氣體荷爾蒙，在植物生長發育過程，扮演著相當多樣的調節角色。過去研究大多著眼於乙烯如何在生物體內自然生成的生化反應機制，及植物在受乙烯刺激後會產生何種生理反應等等，現在則多從分子層級的角度切入，研究乙烯於高等植物中的訊息途徑。目前由阿拉伯芥的研究得知，植物對乙烯的感受性需由特殊的乙烯受器及一連串的訊息傳遞途徑來調控下游受乙烯調控的基因，以達成表型或生理機能的改變，而阿拉伯芥的轉錄調控因子ein3基因產生隱性突變，可以造成乙烯不敏感的性狀。為研究乙烯訊息傳遞的路徑及其相關分子在台灣原生種蝴蝶蘭中表現情形，本實驗以EIN3/EIL基因家族為研究對象，進行蝴蝶蘭PeEIN3 (Phalaenopsis equestris Ethylene-INsensitive3)基因的選殖，研究其構造，並探討其表現情形。首先以EST clone所得到的蝴蝶蘭之乙烯反應轉錄調控因子EIN3基因片段作為探針，進行姬蝴蝶蘭花苞cDNA基因庫篩選，分離出PeEIN3 cDNA共2411 bp (基因索引號AJ316086)，其中開放譯架區為1866 bp可轉譯出621個氨基酸序列，轉譯氨基酸序列80殘基(residues)至300殘基處呈現高保留的特性。序列比對結果，與阿拉伯芥EIN3/EIL基因家族成員具有55-60%的相同度(identity)及68-70%相似度(similarity)。南方墨點分析結果顯示PeEIN3為單一拷貝基因，且其基因族系至少包含二個以上的成員。分子親緣關係之分析顯示PeEIN3在演化上與玉米的ZmEIN3、稻米的OsEIL位於同一個分類枝(clade)，皆屬於單子葉基因群。北方墨點分析PeEIN3基因表現結果顯示，在蝴蝶蘭花苞、花、葉、根PeEIN3基因表現並無明顯的組織特異性。為進一步探討PeEIN3基因的功能是否與阿拉伯芥EIN3相似，故將姬蝴蝶蘭PeEIN3 cDNA利用農桿菌轉殖到阿拉伯芥野生型植株及乙烯不敏感突變植株(ein3-1)，觀察轉殖阿拉伯芥T2世代的小苗發現無論是否有乙烯前驅物ACC的刺激，轉殖株均能持續表現triple response現象，顯示PeEIN3具有與EIN3/EIL基因家族相似的功能。

Ethylene (C2H4) is a gaseous hormone that affects many aspects on plant growth and development. Previous studies focused on the biosynthesis of ethylene and the ability of cells to perceive the hormone and respond in an appropriate manner. At present, the molecular events underlying the ethylene response pathway in higher plants have only begun to be understood. Ethylene perception requires specific receptors and a signal transduction pathway to coordinate down-stream responses. The ein3 gene of Arabidopsis encodes a transcription factor, mutation of ein3 confers recessive ethylene insensitivity. In this study, we cloned and characterized the EIN3 gene of Phalaenopsis equestris. The EIN3 gene fragment from EST clone was used as probe to screen a cDNA library constructed using poly(A)+ RNA from Phalaenopsis equestris flower buds. A cDNA encoding the EIN3 homolog, designated PeEIN3 (Accession No. AJ316086), was obtained and sequenced. The PeEIN3 cDNA is 2411 bp in length, encoding 621 amino acids. The deduced amino acid sequence shares 55-60% identity and 68-70% similarity with Arabidopsis EIN3 and EIN3-Like (EIL) genes. Southern blot analysis indicated that small gene family of EIN3 was present in Phalaenopsis equestris genomes. Molecular phylogenetic analysis of its evolutionary relationship revealed that PeEIN3 is linked to monocot genes, such as ZmEIN3 of maize and OsEIL of rice. Northern blot analysis of Phalaenopsis equestris leaf, root, flower and floral bud indicated that gene expression of PeEIN3 was not tissue-specific. To further explore if the similarity of sequence and expression is coupled to that in function of Arabidopsis EIN3, functional analysis of PeEIN3 through transgenic plants is performed. Sense cDNA of PeEIN3 was ectopically expressed in wild-type and ethylene-insensitive mutant Arabidopsis plants. Four-day-old T2 seedlings generated from both transgenic wild-type and mutant plants all exhibited triple response phenotype in either the presence or the absence of 1-aminocyclopropane- 1-carboxylic acid (ACC), a precursor of ethylene. These observations indicate that PeEIN3 is functionally similar to EIN3/EIL family.

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