文心蘭EIN3同源基因之選殖及啟動子活性分析

Abstract

文心蘭 (Oncidesa spp.) 為臺灣重要外銷切花，花朵及花藥蓋易脫落造成乙烯大量產生而迅速萎凋，為探討乙烯訊息傳導轉錄因子ETHYLENE INSENSITIVE 3 (EIN3)/EIN3-LIKEs (EILs) 基因表現特性，首先選殖文心蘭OgEIL1及OgEIL2，OgEIL1基因結構包含三個顯子及兩個位在5’非轉譯區之隱子，OgEIL2選殖得到轉譯區及轉譯起始點上游2.5 kb序列，兩基因轉譯起始點上游序列經資料庫比對顯示皆具有高溫、光、無氧逆境、激勃素及水楊酸等cis-acting element。進一步將OgEIL1啟動子、5’非轉譯區及隱子構築於含有β-glucuronidase的表達載體，轉殖至阿拉伯芥及菸草，觀察OgEIL1啟動子於各發育階段、生長調節劑及逆境誘導之表現情形。阿拉伯芥OgEIL1pro::GUS包含第一及第二隱子的轉殖株中啟動子表現於頂端分生組織、葉片、根尖、柱頭及花藥，OgEIL1pro::GUS只含第一隱子之轉殖株啟動子表現量較含兩隱子的轉殖株減少，OgEIL1pro::GUS只含第二隱子之轉殖株啟動子只於花瓣表現，未含隱子之OgEIL1pro::GUS轉殖株中啟動子只表現於頂端分生組織、葉片及柱頭，顯示第一隱子具有增強基因表現的功能，且第一及第二隱子皆具有調控啟動子表達部位的功能。IAA及SA可抑制而高溫則誘導OgEIL1pro::GUS同時包含兩個隱子及只含第一隱子的轉殖株之啟動子表現，顯示第一隱子具有對於IAA及SA之silencer且具有對於高溫的enhancer；創傷及乾旱可提升含有兩個隱子、只含第二隱子及無隱子之轉植株啟動子活性，顯示第二隱子具有對創傷及乾旱之enhancer；ABA可抑制含第一隱子轉殖株啟動子活性但提升含第二隱子轉殖株啟動子活性，顯示第一隱子具有對ABA的silencer且第二隱子具有enhancer；ACC、GA及鹽逆境可提升完全不含隱子及同時含二個隱子的轉殖株啟動子活性，但不影響只含單一隱子的轉殖株啟動子表現，顯示隱子中可能並非只有單一因子調控基因表現；MeJA及低溫處理可促進含兩隱子轉殖株的啟動子活性，顯示此兩隱子皆具有對MeJA及低溫的enhancer。四種不同表達載體之菸草轉殖株已經由PCR驗證轉殖株，並由南方氏雜交分析取得單一拷貝之轉殖株。

The cut flower of Oncidesa Gower Ramsey is one of the most important ornamental flowers in Taiwan for exportation. However, the vase life tends to short resulting from the pollinia cap dislodging frequently and production of ethylene. In order to investigate the expression pattern of ETHYLENE INSENSITIVE 3 (EIN3) / EIN3-LIKEs (EILs), the OgEIL1 and OgEIL2 were cloned from Oncidesa. The gene structure of OgEIL1 contains three exon and two intron located in 5’-untranslated region. Several conserved cis-acting elements such as heat, light, anaerobic stress, GA, and MeJA. The OgEIL1 promoter, untranslated region and two introns were fused with β-glucuronidase gene to form different lengths of expression vectors and transformed into Arabidopsis and tobacco via Agrobacterium-mediated method to investigate promoter activity of OgEIL1. The promoter activity was observed by GUS histochemical staining at different development stages, plant growth regulators and stresses treatments. Histochemical staining analysis of transgenic Arabidopsis seedlings carrying OgEIL1pro::GUS containing intron 1 and intron 2 showed that GUS activity was detected in apical meristem, leaves, root tips, stigma and anther. There was low-level GUS activity in Arabidopsis seedlings carrying OgEIL1pro::GUS including intron 1. The GUS activity in Arabidopsis seedlings carrying OgEIL1pro::GUS containing intron 2 was detected only in petals. The GUS activity of Arabidopsis seedlings carrying OgEIL1pro::GUS containing intron 1 was detected in apical meristem, leaves, and stigma, therefore, intron 1 contained enhancer and tissue-specific expression probably is mediated by intron 1 and intron 2. There was lower GUS activity after IAA and SA treatments and higer activity after heat treatment in seedlings carrying OgEIL1pro::GUS including intron 1 or both introns, these results indicated there is a silencer response to IAA and SA and an enhancer response to heat in intron 1. There is a silencer in intron1 and an enhancer in intron2 response to ABA. ACC, GA, and salt increased the promoter expression in seedlings harboring OgEIL1pro::GUS or including both introns and decreased in seedlings harboring OgEIL1pro::GUS including intron 1 or intron 2. These results showed there are complex factors response to these treatments located in intron 1 and intron 2. There are enhancers in both introns response to MeJA and cold. The transgenic tobacco haboring four kinds of expression vectors were confirmed by polymerase chain reaction and Southern analysis for transgenic copy number.