苦瓜EDR1同源基因啟動子活性及CAN1同源基因功能之分析

Abstract

為瞭解苦瓜抗白粉病相關基因McEDR1與核酸分解酶基因McCAN1之基因表現調控情形，本研究將以農桿菌媒介法穩定性轉殖之阿拉伯芥，進行不同發育階段、植物生長調節劑、及非生物性逆境誘導試驗，經GUS活性組織化學染色分析啟動子之活性表現。McEDR1啟動子於阿拉伯芥根尖、下胚軸、生長點、子葉、幼葉、及成熟葉皆有表現。植物生長調節劑IAA、BA、GA3、ABA、MeJA、與SA皆會抑制啟動子之表現，惟有ACC可明顯促進於根尖之活性表現。逆境誘導以高鹽、低溫、及低溫黑暗處理，可提升於新生葉之活性，而以乾燥、高鹽、黑暗、及低溫黑暗處理，根尖亦有明顯之促進效果。McCAN1啟動子表現部位於阿拉伯芥根部、下胚軸、生長點、子葉、幼葉、及成熟葉。植物生長調節劑除SA促進子葉葉脈、及ACC促進地上部葉片之表現，其餘皆抑制啟動子之表現。逆境處理高鹽和低溫黑暗會促進根部之活性表現，而創傷、乾燥、高鹽、低溫、黑暗、及低溫黑暗則會誘導子葉和地上部葉片之啟動子表現。此外，為進一步瞭解McCAN1之基因功能，構築過量表現及默化McCAN1基因、及前後端分別融合綠色螢光蛋白之蛋白質定位用轉殖載體。基因過量表現之構築目前尚未成功獲得菸草及阿拉伯芥轉殖株。基因默化之構築目前已穩定性轉殖於菸草及阿拉伯芥，並以GUS活性組織化學染色及聚合酶連鎖反應確認轉殖株。蛋白質定位將McCAN1暫時性表達於洋蔥表皮細胞及阿拉伯芥原生質體，以共軛焦螢光顯微鏡觀察得知，皆於轉殖後19至22小時表達於細胞之細胞膜上。

The regulation mechanism of McEDR1 and McCAN1 promoters and gene expression analysis of McCAN1 were done in the present study. For promoter analysis, McEDR1 and McCAN1 promoter sequences were constructed to drive b-glucuronidase GUS reporter gene and the promoter activity was observed by GUS histochemical staining at different developmental stages and in response to different plant growth regulators and abiotic stresses in transgenic Arabidopsis pure line. Promoter activity of McEDR1 was expressed in root tips, hypocotyls, shoot meristem, cotyledons, young leaves and mature leaves of Arabidopsis. IAA, BA, GA3, ABA, MeJA, and SA play the inhibitory role on McEDR1 promoter activity. On the contrary, ACC enhanced the promoter activity of McEDR1. High salt, cold, and cold combined with dark treatment enhanced promoter activity in young leaves. Drought, high salt, dark, and cold combined with dark treatment enhanced promoter activity in root tips. McCAN1 promoter was expressed in root tips, hypocotyls, shoot meristem, cotyledons, young leaves and mature leaves of Arabidopsis. All the treated plant growth regulators suppressed promoter activity except SA, which enhanced the activity in veins of cotyledons, and ACC enhanced in leaves. Promoter activity in leaves was enhanced by high salt treatment and cold combined with dark treatment enhanced activity in root. Wound, drought, high salt, cold, dark, and cold combined with dark treatment enhanced promoter activity in leaves. For McCAN1 functional analysis, McCAN1-overexpression plasmid was transformed into tobacco and Arabidopsis and yet to obtain transgenic tobacco or Arabidopsis. Moreover, short interference RNA (siRNA) strategy was used to silence CAN1 genes both in bitter gourd and tobacco. Stable transformation for siRNA construct of 6 lines of tobacco and 3 lines of Arabidopsis were confirmed by GUS histochemical staining and polymerase chain reaction, through the morphological observation of transgenic plants might further elucidate the function of genes. For localization, McCAN1 cDNA was fused with green fluorescent protein (GFP) at front end and rear end, respectively. When co-localized with organelle-specific fluorescent protein markers in onion epidermal cell and Arabidopsis protoplast, the localization of McCAN1 was found on cell membrane.