2012-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/687315摘要：苦瓜 (Momordica charantia L.) 為葫蘆科 (Cucurbitaceae) 一年生蔓性草本植物，是臺灣重要夏季蔬菜之一，兼具食用、藥用與觀賞價值，栽培地區遍及全省，若能生產熱帶亞洲特產、高品質、耐貯運的苦瓜，將可進軍國外市場，具有發展潛力。苦瓜為雌雄異花同株，雄花與雌花比例在7:1～25:1之間，其花性會受到溫度、日長及外施植物生長調節劑等條件而改變。若能探討苦瓜性別變化及其分子機制，將有助於釐清苦瓜性別之決定性因子，應用於提高單株雌花表現及結果數。本研究係延續98年計畫，該計畫選殖得到與花性相關特殊蛋白質脂結合蛋白 (plastid lipid-associated protein, PAP) 之cDNA及基因，並分析其啟動子之活性，故本計畫期望利用過量表現及RNA干擾技術，轉殖PAP基因至苦瓜及胡瓜中，觀察轉殖植株之花性及其他性狀之變化，進一步釐清PAP之功能，以產生雌花提早產生且數目增多之轉殖苦瓜，即可提高苦瓜之產量及品質；另一方面，將質脂結合基因與報導基因綠色螢光蛋白基因融合，除了運用基因槍法於洋蔥表皮細胞及PEG法於阿拉伯芥原生質體進行暫時性表現，另並轉殖至阿拉伯芥及菸草，於穩定性轉殖植株中觀察質脂結合蛋白之作用部位。本計劃將分成下述七大部份來進行： 1.進行苦瓜質脂結合蛋白基因過量表現及基因默化質體之構築 (第一年) 2.進行苦瓜質脂結合蛋白基因融合綠色螢光蛋白基因表現質體之構築 (第一年) 3.苦瓜及胡瓜再生及基因轉殖系統之建立 (第一年) 4.苦瓜質脂結合蛋白定位之暫時性表現分析及穩定性基因轉殖 (第二年) 5.質脂結合蛋白基因表現質體於苦瓜及胡瓜之基因轉殖及篩選 (第二年) 6.苦瓜及胡瓜轉殖植株之分子驗證及性狀分析 (第三年) 7.表現苦瓜質脂結合蛋白基因融合綠色螢光蛋白基因轉殖株之分析 (第三年) 已知苦瓜之PAP基因不具隱子，且其核&#33527;酸序列與胡瓜之質脂結合蛋白同源性高，故可選擇兩個基因相同序列的區域進行基因默化的目標序列，構築為可形成髮夾型 (hairpin) 之表現質體，轉殖至苦瓜及胡瓜。綠色螢光蛋白基因則將融合於PAP基因下游，與帶不同報導基因之胞器標誌質體共轉殖後，將以共軛焦顯微鏡進行觀察，釐清其蛋白質累積部位。<br> Abstract: Bitter gourd is one of the typical tropical vegetables originated from Taiwan. It has great economic importance in agriculture with edible, medicinal, and ornamental value. The proportion between staminate (male) and pistillate (female) flowers in monoecious plants is 7:1 to 25:1. Sex expression of bitter gourd can be regulated by application of various growth regulators such as GA3, auxin, Ethrel, morphactin. Sex expression in bitter gourd is also influenced by environmental factors such as vernalization and day length. The cDNA and gene coding for sex-related plastid lipid-associated protein (PAP) have been isolated and characterized from bitter gourd in my last grant program. This proposal is intended to overexpress and suppress PAP gene in bitter gourd and cucumber for functional analysis. Furthermore, PAP gene fused with green fluorescent protein gene will be introduced into epidermis of onion and protoplasts of Arabidopsis via transient expression system for protein localization. Agrobacterium-mediated transformation will also be performed for stable expression. According to the research items, this proposal can be divided into the following seven parts: 1. Construction of overexpression and RNA interference plasmids for PAP gene (The first year) 2. Construction of PAP gene fused with green fluorescent protein gene (The first year) 3. Establishment of transformation systems for bitter gourd and cucumber (The first year) 4. Transient assay and stable transformation of protein localization for PAP (The second year) 5. Transformation of bitter gourd and cucumber and antibiotic selection for transgenic plants. (The second year) 6. Molecular identification for transgenic plants and observation for phenotype of transgenic plants (The third year) 7. Protein localization for PAP in transgenic plants via confocal microscopy (The third year) PAP gene is without intron interruption in bitter gourd and shares high sequence homology with PAP from cucumber. Green fluorescent protein will be fused with PAP C-terminus. Transgenic plants will be examined for protein localization under confocal microscopy and phenotype change.性別決定綠色螢光蛋白過量表現RNA干擾技術基因轉殖Sexual determinationGreen fluorescence proteinoverexpressionRNA interferencegenetic transformation