乙烯(C2H4)為氣體形式作用的植物荷爾蒙，參與許多植物的生長發育與生物、非生物逆境反應。乙烯和乙烯受器結合後之訊息藉由MAPK 途徑進行傳遞，乙烯受器為訊息進入細胞的最上游。典型的乙烯受器由3個功能區組成：乙烯結合區、組胺酸激酶功能區及接收功能區。乙烯受器具有2種型式：ETR型態之乙烯受器具有C端之接收功能區，ERS型態則無。蝴蝶蘭為重要經濟花卉之一，台灣原生種姬蝴蝶蘭(Phalaenopsis equestris)為紅花重要親本之一，研究乙烯之訊息傳遞相關分子將有助於產業應用。本實驗以乙烯受器基因為研究對象，進行姬蝴蝶蘭乙烯受器基因之選殖與特性分析。首先由基因資料庫中其它植物物種之乙烯受器保留性區域設計簡併性引子(degenerate primer)，擴增姬蝴蝶蘭乙烯受器基因片段並選殖定序，由基因片段進行5´-及3´-RACE得到完整之姬蝴蝶蘭乙烯受器基因全長，命名為PeERS，登錄序號為AJ563284。PeERS基因全長共有2,025 bp，譯碼區有1,092 bp，轉譯出633個胺基酸，推測其分子量為71 kDa，等電點為6.61。比對分析胺基酸序列後，發現PeERS具有3個穿膜區及組胺酸激酶保守區，並不具有C端訊息接收區，因此其屬於ERS型態之乙烯受器。PeERS胺基酸序列在第4、6個胺基酸具有保守之cysteine，為乙烯受器形成二聚體相關之重要胺基酸。利用南方墨點法分析姬蝴蝶蘭乙烯受器基因族系之組成，以PeERS基因譯碼區為探針，至少出現2個雜合反應條帶，推測姬蝴蝶蘭基因組具有2個乙烯受器基因族系成員；以近3´非譯碼區為探針，僅出現1個雜合反應條帶，因此推測PeERS為單一拷貝基因。利用北方墨點法分析PeERS基因表現，顯示PeERS在根、葉及花均有表現，但在老葉的表現量最高，幼苗的表現量最低，因此PeERS可能與器官之老化相關。將姬蝴蝶蘭去花粉蓋1天，發現PeERS並未因為去花粉蓋而表現量增加，顯示PeERS基因表現並不會受到內生性乙烯增加所誘導，推測姬蝴蝶蘭去雄凋萎是PeERS經由後轉錄作用進行調控。利用聚類分析法構築不同物種之乙烯受器基因演化樹，顯示PeERS位於單子葉植物分類枝上。親源演化樹在一開始分做兩群，兩群均含有單子葉及雙子葉植物，推測乙烯受器基因的分歧是在單雙子葉分化之前。再以轉基因植物檢測PeERS基因功能，利用花椰菜鑲嵌病毒35S啟動子持續表現姬蝴蝶蘭PeERS基因，轉殖到野生型及乙烯不敏感突變株(etr1-1)阿拉伯芥，T2世代轉殖植株並未呈現明顯三相反應表型，推測轉殖PeERS基因並未在阿拉伯芥內表現功能，原因需進一步探討。

Ethylene is a gaseous phytohormone, which regulates many aspects of plant growth and development as well as in response to biotic and abiotic stresses. Ethylene/receptor binding leads to a plant response through the ethylene MAPK signal transduction pathway. Therefore, ethylene receptor is the early element of this pathway. Typical ethylene receptor protein exists as a membrane-associated dimer and has three domains: amino-terminal ethylene binding domain, histidine kinase domain and receiver domain. Two classes of ethylene receptors, ETR- and ERS-type, the latter is devoid of receiver domain. Phalaenopsis orchid is one of the most commercially important floral crops in Taiwan. Phalaenopsis equestris, a wild species orchid, native to Taiwan and widely used as parental strains for breeding red flower orchid cultivars. Studying of ethylene signaling molecules in orchids may provide useful information for floriculture industry applications. Therefore, the objectives of this study were cloning and analysis of a cDNA coding for ethylene receptor from Phalaenopsis equestris. Firstly, the degenerate primers on the basis of ethylene receptor conserved domain from different plant species were designed. The cDNA encoding a ethylene receptor of Phalaenopsis equestris was obtained by a combination of RT-PCR and 5´-/3´-RACE cloning. This Phalaenopsis equestris ethylene receptor was named as PeERS (Accession No. AJ563284). The cloned cDNA is 2,025 nucleotides long and the deduced amino acid sequence comprised 633 amino acid residuces with a caculated molecular mass of 71 kDa, isoelectric point (pI) of 6.61. The amino acid sequence of PeERS contained all the conserved residues of the two domains, a transmembrane domain and a conserved histidine kinase domain, but lacked a receiver domain. Thus, PeERS belongs to an ERS-type. Two cysteine residues that are required for dimerization are also found in PeERS receptor at position 4 and 6. Southern blotting of genomic DNA with PeERS coding region suggested that there were at least two members in Phalaenopsis equestris ethylene receptor gene family. Using 3´UTR as probe showed that PeERS was a single-copy gene. Northern analysis showed that PeERS was expressed in all organs examined, i.e. root, leaf and flower. High relative amounts were detected in the old leaf in comparison with the seedling, suggesting that PeERS may involve in the organ senescence. The expression pattern of PeERS after emasculation did not change significantly, indicating that PeERS could not be induced by endogenous ethylene. It is possible that ethylene sensitivity in Phalaenopsis equestris flowers might be regulated at the post transcriptional level. A phylogenetic tree was constructed by Neighbor-Joining method, demonstrating that PeERS clustered within the monocot ethylene receptor gene clade. This phylogenetic tree divided into two clades and for each of clade consists of both monocots and dicots, suggesting that ethylene receptor genes were present before the divergence of monocot and dicot plants. Furthermore, the function of PeERS was also analyzed using transgenic plant approach. Sense cDNA of PeERS under control of the constitutive CaMV35S promoter was introduced into wild type and ethylene insensitive mutants (etr1-1) Arabidopsis plants. T2 seedlings generated from transgenic plants did not reveal marked triple response phenotypes. The results suggested that the PeERS transgene was not functionally active in the transgenic Arabidopsis plants. The causes for these results need to be explored by further experiments.

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