苦瓜與蝴蝶蘭 EIN3 同源基因之啟動子活性研究

Abstract

乙烯為氣態植物荷爾蒙，於作物發育和逆境反應具重要調控功能，已知阿拉伯芥 ETHYLENE INSENSITIVE3 (EIN3) 與 EIN3-like 1 (EIL1) 轉錄因子是乙烯訊息傳導途徑調節關鍵因子。為了瞭解更年性作物苦瓜和蝴蝶蘭之 EIN3 同源基因啟動子活性及第一個隱子之重要性，分別將苦瓜 McEIL1 和 蝴蝶蘭 PtEIL1 之啟動子序列連結報導基因 GUS，並構築含或不含5’ 不轉譯區 (5’ untranslated region, 5’-UTR) 內隱子之不同轉殖質體，以農桿菌轉殖至菸草。McEIL1pro::GUS 菸草轉殖株於發芽時胚根和上胚軸皆明顯表現；播種後 10-15 天幼苗於莖與根部皆表現，於莖頂表現明顯；播種後 20-30 天，根、莖與莖頂之表現隨幼苗年齡增加而逐漸增強，子葉與下位葉之葉脈和葉肉之表現亦逐漸增強，年齡較低之葉片表現量較低；於成熟株葉片、花瓣、花萼、柱頭、花藥、果實之果皮、胎座和胚珠皆表現。McEIL1pro::GUS 菸草轉殖株於播種後 15 天幼苗可受到 4℃、37 ℃、黑暗環境、創傷、IAA、GA3、ABA、SA 處理之誘導，使表現量增加；以 BA 和 ACC 處理，表現量變化不明顯。McEIL1pro::GUS 含 5’-UTR隱子之菸草轉殖株表現量較不含 5’-UTR 隱子高，推測此隱子具有 suppressor 而抑制 McEIL1 啟動子活性。 PtEIL1 mRNA 於蝴蝶蘭根、莖、葉與花器皆表現，相對表現量於葉片最高，根部次之，接著為莖部，於開展但未老化之花器表現量最低；花器分為翼瓣、唇瓣和蕊柱，其中於蕊柱之表現量較高。PtEIL1pro::GUS 不含 5’-UTR 隱子之菸草轉殖株於發芽時於胚根表現，但表現量不高；播種後 0-15 天幼苗於莖頂表現量較明顯；播種後 25-30 天幼苗之子葉與下位葉之表現量隨年齡增加而逐漸增強；於成熟株葉片、花瓣、花萼、柱頭和花藥均表現，於果實表現量低。PtEIL1pro::GUS不含 5’-UTR 隱子之菸草轉殖株於播種後 10 天幼苗可受到 4℃ 、37 ℃、黑暗、淹水逆境、GA3、ABA、SA 處理誘導，使表現量增加；以 IAA、BA 和 ACC 處理，表現量變化不明顯。PtEIL1pro::GUS 含 5’-UTR 隱子之菸草轉殖株於幼苗皆不表現，即使以非生物逆境或生長調節物質誘導亦不表現，於成熟葉片和花器之表現位置與 PtEIL1pro::GUS 不含 5’-UTR 隱子之菸草轉殖株相似，推測其 5’-UTR 隱子亦具有抑制啟動子活性之機制，於幼苗之表現受抑制或不表現。

Ethylene, a gas hormone in higher plants, plays an important role in crop development and stress responses. EIN3 and EIL1 transcription factors found in Arabidopsis were known as critical regulating factors on ethylene signaling transduction. EIN3 homologous genes, McEIL1 from climate crop bitter gourd; and PtEIL1 from Phalaenopsis, were cloned to investigate the promoters activity and the role of the first intron in 5’-UTR. 5’-upstream sequences of both genes with or without the first introns were fused with GUS and transformed into tobacco via Agrobacterium-mediated methods. Promoter activity of McEIL1pro::GUS transgenic tobaccos expressed at seed germination and five-day old seedlings, especially at radicals and epicotyls. Ten-day old and fifteen-day old seedlings showed expression at roots and stem, the shoot tip showed strong activity. The promoter activity of ten-day old to fifteen-day old seedlings gradually increased at shoot tip, cotyledon, veins and mesophyll of lower position leaves. Mature plants of McEIL1pro::GUS transgenic tobaccos showed expression at leaves, petals, sepals, stigma, and anther; fruits showed expression at placenta and ovules. 15-day old McEIL1pro::GUS transgenic tobacco seedlings were induced by treating with 4℃, 37 ℃, darkness, wounding, IAA, GA3, ABA, and SA. BA and ACC treatments make no difference to the McEIL1 promoter expression. The McEIL1pro::GUS expression were reduced when the McEIL1 promoter construction contains the 5’-UTR intron. This suggested a suppressor in the intron may participate in promoter expression of McEIL1. PtEIL1 mRNA accumulated in roots, stem, leaves, and flowers of Phalaenopsis. Leaves showed highest expression, followed by stem and roots. The fully open flower showed lower expression compared with the vegetative organs. The flower were separated to petals, labellum, and gynostemium, and the PtEIL1 mRNA expressed higher at gynostemium. The transgenic tobaccos of PtEIL1pro::GUS containing the 5’-UTR intron showed promoter activity at germinating seeds and shoot tip of 5-day old to 15-day old seedlings. However, the expression level were low. 25-day old to 30-day old seedlings gradually increased at veins and mesophyll of lower position leaves. Mature plants of PtEIL1pro::GUS transgenic tobaccos showed expression at leaves, petals, sepals, stigma, and anther, but not at fruits. The promoter activity of transgenic tobacco seedlings of PtEIL1pro::GUS containing the 5’-UTR intron were induced by 4℃, 37 ℃, darkness, wounding, flooding, GA3, ABA, and SA treatments. IAA, BA and ACC treatments make no difference to the PtEIL1 promoter expression. The transgenic tobacco showed no PtEIL1pro::GUS expression at seedlings when the PtEIL1 promoter construction contains the 5’-UTR intron, even after treated with plant growth regulator and abiotic stresses. However, the leaves and flower from mature plants showed expression. This suggested the intron may participate in a inhibitor mechanism to change the promoter activity level or the location of expression.