苦瓜質脂結合蛋白cDNA之選殖與分析

Abstract

苦瓜 (Momordica charantia L.) 為雌雄異花同株之葫蘆科作物，其產生雄花及雌花之比例可利用外施植物生長調節劑加以調節。經以蛋白質體學試驗，顯示質脂結合蛋白 (plastid-lipid associated protein, PAP) 在雌花發育階段有特異表現。為瞭解質脂結合蛋白對花性決定之機制，以阿拉伯芥質脂結合蛋白基因為探針，進行溶斑形成雜交法，篩選月華苦瓜cDNA庫一百五十萬個溶斑形成單位 (plaque forming unit, pfu)，得到36個選殖系。其中pMcPAP-76 cDNA長1,226 bp，包含一個長984 bp之開放閱讀框架 (open reading frame)，可演繹出327個胺基酸，預測之分子量與等電點分別為35.53 kDa及4.89，將其基因命名為McPAP1。McPAP1胺基酸序列和其他植物物之PAP同源性界於62.7~79.9%，與胡瓜雜色體蛋白C (chromoplast protein C, CHRC) 之相似性最高為79.9%。由南方氏雜交分析，得知McPAP1在月華苦瓜基因組中為單一拷貝基因。分析McPAP1之基因表現，顯示其在盛開之雄花、雌花及果實有專一性表現，在花器表現上，McPAP1在盛開的雄花表現量最大，而在早期發育之雌花表現量較高。偵測McPAP1於不同授粉天數果實中表現情形，顯示隨著果實的發育，基因表現增加，於授粉後第18天表現大量增加，至授粉後第24天之發育後期，達到最大之表現量，約為授粉後第18天之1.5倍。為了瞭解植物生長調節劑對McPAP1基因表現之影響，分別以IAA及乙烯處理果實，北方雜交分析結果顯示其基因表現會受到乙烯誘導而受到IAA抑制。為釐清McPAP1表達位置及與性別表現之相關性，將進行GFP融合蛋白載體之暫時性表達分析。

Bitter gourd (Momordica charantia L.) is a monoecious species in Cucurbitaceae. Sex expression of bitter gourd is altered by exogenous application of plant growth regulators. Using 2- dimensional protein gel electrophoresis to identify sex expression-related proteins in bitter gourd, the results indicated that the plastid-lipid associated protein (PAP) expressed specifically during female flower development. To understand the mechanism of sex determination, plaque hybridization was performed to screen the PAP cDNA from bitter gourd library using Arabidopsis thaliana PAP gene as probe. Thirty-six cDNA clones were obtained. cDNA clone pMcPAP76 is 1,226 bp in length and possesses an open reading frame of 984 bp, enconding a polypeptide of 327 amino acids with an isoelectric point of 4.89 and a molecular weight of 35.53 kDa. The corresponding gene of pMcPAP76 cDNA is named McPAP1. The deduced amino acid sequence of McPAP1 shared high homology with other PAP around 62.6% to 79.9%, especially cucumber chromoplast protein C (CHRC) with 79.9% identity. Southern blot analysis indicated that McPAP1 belongs to a single-copy gene. Northern blot and RT-PCR analysis reflected that the expression of McPAP1 was specific in flower and fruit. The McPAP1 gene expressed most abundantly in male flowers at full blussom, but more abundantly at early stage in female flowers. The McPAP1 gene expression was consisted in the fruit development stage. The McPAP1 gene expression in fruit was found largest at 24-day after pollination which was 1.5 times as much as did that at 18-day after pollination. McPAP1 expression was induced in fruit tissues treated with ethylene, but inhibited by IAA. To clarify the McPAP1 localization and the relation to sex expression, GFP-fusion protein was constructed for transient analysis.