苦瓜植物生長素及乙烯受體基因之啟動子活性分析

Promoter activity analysis of auxin and ethylene receptor genes from bitter gourd (Momordica charantia L.)

林政谷(Cheng-Ku Lin)

指導教授 ： 杜宜殷

共同指導教授 ： 黃鵬林(Pung-Ling Huang)

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10573

摘要

苦瓜 (Momordica charantia L.) 是臺灣夏季主要蔬菜作物之一，為瞭解植物生長素及乙烯對苦瓜果實發育及後熟之影響。本論文針對苦瓜植物生長素受體基因McTIR1 (TRANSPORT INHIBITOR RESPONSE 1)、乙烯受體基因McETR1 (ETHYLENE RECEPTOR 1)及McERS1 (ETHYLENE RESPONSE SENSOR 1) 進行啟動子活性分析，於菸草及阿拉伯芥中觀察上述啟動子於各發育階段、逆境及生長調節劑誘導的基因表現情形。McTIR1啟動子於阿拉伯芥及菸草轉殖株之花萼、雄蕊和雌蕊等組織皆有表現；於菸草種皮、下胚軸、葉、莖頂、根尖及花瓣等其他部位亦可偵測到基因之表現。經創傷及黑暗培養處理，結果顯示啟動子活性可於轉殖株葉片及莖部受到誘導促進表現，而4°C低溫及乾旱逆境則會部分抑制啟動子活性於葉片之表現。處理ABA及不同種類植物生長素後，可部分抑制葉片中McTIR1啟動子活性，其中以ABA抑制效果較為顯著。另一方面，經分析基因組選殖系，取得苦瓜乙烯受體基因McETR1序列9,213 bp及McERS1序列7,313 bp，分析兩基因基因結構皆由7個顯子及6個隱子組成，啟動子序列分析結果顯示，兩啟動子皆含ABRE (ABA responsive element)、GARE (GA responsive element)、HSE (Heat stress responsiveness element)、CGCTA (MeJA- responsiveness) 及光反應序列等調控因子。McETR1啟動子於菸草轉殖株之分子驗證已完成，待取得菸草之轉殖純系種子。McETR1啟動子活性於阿拉伯芥子葉、莖部節間、側根、根尖、雄蕊、雌蕊及果莢表現。經不同植物生長調節劑處理阿拉伯芥轉殖株，ACC、IAA、GA和SA可誘導苦瓜乙烯受體基因McETR1啟動子於阿拉伯芥主根、側根及莖段連結節間處表現，以ACC處理誘導效果最佳。

關鍵字

苦瓜 ； 植物生長素 ； 乙烯 ； 受體基因 ； 啟動子活性分析

並列摘要

The bitter gourd (Momordica charantia L.) is one of prominent summer vegetable crop in Taiwan. In order to understand influence of auxin and ethylene on fruit development and ripening for bitter gourd, auxin receptor McTIR1 (TRANSPORT INHIBITOR RESPONSE 1) and ethylene receptor genes McETR1 (ETHYLENE RECEPTOR 1) and McERS1 (ETHYLENE RESPONSE SENSOR 1) from bitter gourd were analyzed for promoter activity in tobacco and Arabidopsis at different developmental stages and treatments with abiotic stresses and plant growth regulators. Promoter activity of McTIR1 was expressed in calyx, pistil and stamen of McTIR1pro::GUS transgenic Arabidopsis and tobacco, which also expressed in seed coat, hypocotyls, leaf, stem, shoot, root tip and petal of transgenic tobacco. The promoter activity was enhanced in leaf and stem of transgenic tobacco seedlings by wounding or under dark, but suppressed in leaf by 4oC and drought. Both ABA and auxins play the inhibitory role on McTIR1 promoter activity in leaf of transgenic tobacco seedlings, while the manifest inhibitory effect was contributed by ABA. On the other hand, 9,213 bp of McETR1 and 7,313 bp of McERS1 were obtained from genomic clones for ethylene receptor gene structure and promoter sequence. Both ethylene receptor genes from bitter gourd possess seven exons and six introns and several cis-acting elements, such as ABRE (ABA responsive element)、GARE (GA responsive element)、HSE (Heat stress responsiveness element) and CGCTA (MeJA-responsiveness). Gene integrity and copy number for McETR1::GUS transgenic tobacco has been analyzed by Southern hybridization. McETR1 promoter was expressed in cotyledon, stem internode, later root, root tip, pistil, stamen and siliques of transgenic Arabidopsis. When treated by ACC, IAA, GA, or SA, the promoter activity in McETR1::GUS transgenic seedling was enhanced in main root, later root, and stem internode with most obvious effect by ACC.

並列關鍵字

bitter gourd ； auxin ； ethylene ； receptor ； promoter activity

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