苦瓜質脂結合蛋白基因 McPAP1 之啟動子活性分析

Promoter Activity Analysis of McPAP1 Encoding a Plastid lipid-Associated Protein in Bitter Gourd (Momordica charantia L.)

王瑞婷(Ruei-Ting Wang)

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(2010年)

https://doi.org/10.6342/NTU.2010.10237

摘要

為了瞭解苦瓜質脂結合蛋白 (Plastid lipid-associated protein, PAP) McPAP1 基因表現調控情形，本研究取得 McPAP1 基因及啟動子序列共5,731 bp，構築包含轉譯起始點前 2,859 bp序列之 McPAP1Pro::GUS 之表現載體，以基因槍法轉殖至蝴蝶蘭，及農桿菌媒介法穩定性轉殖於菸草及阿拉伯芥中，並以 GUS 活性組織化學染色法觀察不同生長階段及誘導試驗之活性表現。暫時性表現分析顯示McPAP1 啟動子可於異源白花蝴蝶蘭花瓣表現活性，而阿拉伯芥轉殖株於幼葉、葉基部、老葉、花器及果莢大量表現活性，其中花器的萼片、花瓣、柱頭及雄蕊等多處皆有表現，顯示 McPAP1 可能參與植株生長發育過程多個部份。誘導試驗結果顯示，McPAP1 啟動子於地上部活性表現可受到 BA、MeJA、ABA 及 ACC 誘導促進表現，SA、GA3 處理則抑制表現，IAA 於地上部的誘導效果不明顯，但明顯促進 McPAP1 在根部的表現；光線因子負向調控 McPAP1 之啟動子活性，而溫度逆境則為正向調控，且影響層次位於光線因子之上，另外，高鹽逆境也促進 McPAP1 基因表現，但淹水及乾旱等水份逆境則輕微抑制基因於地上部之表現。阿拉伯芥轉殖株地上部與根部之誘導結果存在差異性，顯示 McPAP1 基因表現在地上部與根部調控機制可能不同，可再進行菸草轉殖株分析以比較結果。

關鍵字

質脂結合蛋白 ； 啟動子 ； 發育階段 ； 植物生長調節劑 ； 逆境

並列摘要

To understand the regulation mechanism of McPAP1 gene expression, 5,731 bp McPAP1-containing fragment was sequenced and 2,859 bp fragment upstream of the ATG start codon was constructed to drive β-glucuronidase reporter gene (McPAP1Pro::GUS) for analysis of promoter activity. The plasmid was transformed into white Phalaenopsis petal by particle bombardment and tobacco and Arabidopsis by Agrobacterium- mediated method and promoter activity was observed by GUS histochemical staining at different development stages. Transient analysis showed McPAP1 promoter activity in white Phalaenopsis petal and transgenic Arabidopsis plant showed GUS activity in young leaf, leaf base, old leaf, flower and siliques, including sepal, petal, stigma and stamen in flower. It was suggested that McPAP1 may participate in lots of different developmental stages. Results of plant growth regulator treatments indicated that BA, MeJA, ABA and ACC induced McPAP1 transcription on upperground part of plant, SA and GA3 reduced the gene expression and IAA specifically increased transcription in root but with no influence on upperground part. According to the results of stress treatment, light played a down regulation role to McPAP1 transcription, but temperature stress upregulated the transcription of McPAP1 and upstream to light. Moreover, salt stress also increased but flooding and drought slighty inhibited transcription level of McPAP1 on upperground part of plant. The differential expression of McPAP1 between upperground part and root, could be examined in transgenic tobacco.

並列關鍵字

Plastid lipid-associated protein ； promoter ； developmental stage ； plant growth regulator ； stress

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