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研究生:林家弘
研究生(外文):Chia-hung Lin
論文名稱:轉殖阿拉伯芥植株外源表現甘藷cysteineproteaseSPCP2促進早開花及增加乾旱耐性
論文名稱(外文):Ectopic expression of sweet potato cysteine protease SPCP2 promotes earlier flowering and enhances drought stress tolerance.
指導教授:陳顯榮陳顯榮引用關係
指導教授(外文):Hsien-Jung Chen
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:60
中文關鍵詞:半胱胺酸蛋白質分解酵素甘藷
外文關鍵詞:cysteine proteasesweet potato
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SPCP2是由甘藷老化葉片分離出來的全長cDNA,藉由氨基酸序列比對與papain-like 半胱胺酸蛋白質分解酵素(papain-like cysteine protease) 類似。SPCP2 ORF(open reading frame)含有1101個核苷酸(366個氨基酸),與奇異果(Actinidia deliciosa)、阿拉伯芥(Arabidopsis thaliana)、花椰菜(Brassica oleracea)、菜豆(Phaseolus vulgaris)、 野豌豆(Pisum sativa)、蠶豆(Vicia faba)、巢菜(Vicia sativa) 及 豇豆(Vigna mungo)等植物的半胱胺酸蛋白質分解酵素具有高度相似性(68%~83%)。SPCP2基因表現顯著增加於甘藷自然老化葉片及已萌芽的貯存塊根。利用轉殖系統將SPCP2外源表現於阿拉伯芥植株進行分析,發現外源表現SPCP2基因可促進轉殖阿拉伯芥植株提早開花時間、減少開花時rosette leaves葉片數目、增加轉殖植株種子發芽時之耐鹽性、乾旱處理時轉殖植株具有較高的光合作用效率 (Fv/Fm value) 、較高的相對水份含量及增加轉殖植株之耐旱性。根據上述結果結論甘藷papain-like半胱胺酸蛋白質分解酵素SPCP2是一個具有功能性的基因,外源表現甘藷SPCP2會改變轉殖阿拉伯芥植株生長發育特徵及耐乾旱與耐鹽分的反應。
Sweet potato SPCP2 is a full-length cDNA isolated from senescent leaves and encodes a putative papain-like cysteine protease. The SPCP2 contained 1101 nucleotides (366 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 68% to 83%) with plant cysteine proteases, including Actinidia deliciosa, Arabidopsis thaliana, Brassica oleracea, Phaseolus vulgaris, Pisum sativa, Vicia faba, Vicia sativa and Vigna mungo. SPCP2 gene expression was enhanced significantly in natural senescent leaves and in sprouting storage root. Transgenic Arabidopsis plants with ectopic constitutive SPCP2 expression showed earlier floral transition from vegetative to reproductive growth, reduced rosette leaves when flowering, enhanced germination percentage of transgenic progeny seeds in salt-containing MS medium, higher Fv/Fm value, higher relative water content and enhanced tolerance during drought treatment. Based on these results, we conclude that sweet potato papain-like cysteine protease, SPCP2, is a functional gene, and its expression causes altered developmental characteristics and enhances drought and salt stress responses in transgenic Arabidopsis plants.
第一章、前言…………………………………………………1
第一節 甘藷……………………………………………1
第二節 葉片老化的生理………………………………2
第三節 半胱胺酸蛋白質分解酵素的研究……………5
第四節 研究方向………………………………………8
第二章、材料與方法…………………………………………9
第一節 實驗材料………………………………………9
一、甘藷(Ipomoea batatas)………………………9
二、SPCP2 cDNA……………………………………9
三、SPCP2轉殖阿拉伯芥種子………………………10
四、轉殖阿拉伯芥植株的種植………………………10
第二節 實驗方法……………………………………11
一、甘藷塊根SPCP2基因表現的檢測……………11
二、對外源表現 SPCP2 轉殖植株生長發育性狀影響分析…………………………………………14
三、外源表現SPCP2對轉殖植株種子發芽於鹽分逆境之影響分析……………………………………14
四、外源表現 SPCP2 對轉殖植株乾旱逆境之影響分析………………………………………………15
五、分析SPCP2之Putative catalytic domain area 及Phylogenetic tree……………………………16
第三章、結果…………………………………………………18
第一節 甘藷自然老化葉片及已萌芽的貯存塊根顯著增加SPCP2基因表現……………………………18
第二節 外源表現SPCP2基因可促進轉殖阿拉伯芥植株提早開花………………………………………18
第三節 外源表現SPCP2基因可增加轉殖阿拉伯芥植株種子發芽之耐鹽性……………………………19
第四節 外源表現SPCP2基因可增加轉殖阿拉伯芥植株之耐旱性………………………………………20
第五節 甘藷SPCP2基因之Putative catalytic domain area 及 Phylogenetic tree analysis.……………………………………21
第四章、討論…………………………………………………23
第五章、參考文獻……………………………………………29
圖…………………………………………………………42

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