苦瓜質脂結合蛋白 McPAP1 之功能分析及蛋白質定位

Functional Analysis and Protein Localization of Plastid Lipid-Associated Protein McPAP1 from Bitter Gourd (Momordica charantia L.)

游沐慈(Mu-Tzu Yu)

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.2010.10746

摘要

已知質脂結合蛋白 (plastid lipid-associated protein, PAP) 為雌雄異花同株的苦瓜 (Momordica charantia L.) 之花性相關蛋白質之一，基因名為McPAP1；為瞭解此蛋白之作用部位及功能，首先 McPAP1 cDNA 序列融合綠色螢光蛋白(green fluorescent protein, GFP)，與螢光蛋白分子標誌共同轉殖至洋蔥 (Allium cepa) 表皮細胞及阿拉伯芥 (Arabidopsis thaliana) 原生質體，確認 McPAP1 位於質粒內；更進一步將 2xCaMV 35Spro::McPAP1:GFP 載體穩定性轉殖至菸草，取得穩定表現 McPAP1:GFP 之菸草轉殖株，以供未來可更進一步研究 McPAP1 於細胞內後轉譯修飾及細胞定位機制。基因功能方面，過量表現 McPAP1 之菸草 (Nicotiana tabacum L. ‘W38’) 轉殖株於光週 16/8 小時、光強度 300 μmol m-2 s-1 之環境下生長，具有提早開花、側芽發育不顯著以及葉基肥大之特性，而分析其葉片色素含量結果顯示，轉殖株之總類胡蘿蔔素含量略高於未轉殖株；進一步以反轉錄聚合酶連鎖反應 (reverse transcription polymerase chain reaction, RT-PCR) 偵測轉殖株內類胡蘿蔔素生合成途徑關鍵酵素之基因表現量變化，以八氫茄紅素合成酶 (phytoene synthase) 及胡蘿蔔素異構酶 (carotene isomerase) 二基因之mRNA 累積量增加最為顯著，顯示 McPAP1 之表達對類胡蘿蔔素生合成途徑具正向回饋效應。而以 short interference RNA (siRNA) 策略構築可同時默化苦瓜 McPAP1 及胡瓜 CHRC 基因之質體，利用阿拉伯芥原生質體進行基因默化效力測試，結果顯示此默化質體於轉殖後 36 小時之默化效力為 32%。

關鍵字

質體小球 ； 胞器專一螢光蛋白分子標誌 ； 類胡蘿蔔素生合成途徑 ； 提早開花 ； 側芽

並列摘要

Plastid lipid-associated protein (PAP) is one of the sexual specific-expressed proteins in monoecious bitter gourd (Momordica charantia L.) and is coded by McPAP1 gene. To determine the localization of McPAP1, the cDNA was fused with green fluorescent protein (GFP) gene and then co-localized with organelle-specific fluorescent protein markers in onion (Allium cepa) epidermal cells and Arabidopsis thaliana protoplasts. Both results indicated that McPAP1 exists in plastids. Furthermore, the construct of 2xCaMV 35Spro::McPAP1:GFP was transformed via Agrobacterium into tobacco (Nicotiana tabacum ‘W38’) and might served as a material for the future researches in posttranslational modification and ultrastructure localization mechanism of McPAP1. For functional analysis, McPAP1-overexpressed plasmid was also transformed into tobacco. The McPAP1-overexpressed tobaccos showed early flowering, less branching and a wider leaf base in the environment with 16/8 photoperiod and 300 μmol m-2 s-1 light intensity. Moreover, pigment content assay exhibits that McPAP1-overexpressed plants contained higher total carotenoid contents than untransformed tobacco did. Gene expressions of carotenoid biosynthetic key enzymes were further examined by reverse transcription polymerase chain reaction. It was shown that the expression levels of those enzymes, PHYTOENE SYNTHASE and CAROTENE ISOMERASE, were enhanced by overexpressed McPAP1. Besides, short interference RNA (siRNA) strategy was used to silence PAP genes both in bitter gourd and cucumber (Cucumis sativus L.). The silencing efficiency was examined by co-transformed McPAP1:GFP-overexpression plasmid and siRNA plasmid into Arabidopsis protoplasts. The silencing efficiency reached 32% at 36 hours after transformation.

並列關鍵字

plastoglobule ； organelle specific fluorescent marker ； carotenoid biosynthesis pathway ； early flowering ； shoot branching

參考文獻

江筱慧. 2008. 苦瓜質脂結合蛋白cDNA之選殖與分析. 國立台灣大學園藝學系碩士論文.

王瑞婷. 2010. 苦瓜質脂結合蛋白基因 McPAP1 之啟動子活性分析. 國立台灣大學園藝學系碩士論文.

Auldridge, M. E., D. R. McCarty, and H. J. Klee. 2006. Plant carotenoid cleavage oxygenases and their apocarotenoid products. Curr. Opi. Plant Biol. 9:315-321.

Austin II, J. R., E. Frost, P. Vidi, F. Kessler, and L. A. Staehelina. 2006. Plastoglobules are lipoprotein subcompartments of the chloroplast that are permanently coupled to thylakoid membranes and contain biosynthetic enzymes. Plant Cell 18:1693-1703.

Bartley, G. E., P. A. Scolnik, and P. Beyer. 1999. Two Arabidopsis thaliana carotene desaturases, phytoene desaturase and zetacarotene desaturase, expressed in Escherichia coli, catalyze a poly-cis pathway to yield pro-lycopene. Eur. J. Biochem. FEMS 259:396–403.