臺灣博碩士論文加值系統

葉片老化為葉片發育的最後階段。在這個階段，老化的葉片會將可回收的養分轉運至它處，以供再利用。葉片老化除了已知需核基因的參與，越來越多的證據顯示這個過程是一種細胞計劃性死亡(programmed cell death, PCD)。目前植物學家紛紛以分子生物的方法來尋找可能參與葉片老化的基因。本研究採用蛋白質二維電泳及mRNA differential display來尋找甘藷葉片自然老化時表現的蛋白質及基因。蛋白質二維電泳分析結果顯示，四個蛋白質特定在老化的葉片中出現，且有再現性，依分子量命名為Sp30-1、Sp30-2、Sp43及Sp45。經製備性電泳取得足夠量之蛋白質進行胺基端定序，接著將序列進行資料庫比對得知，Sp30-1和Sp30-2均為cathepsin類的蛋白質，而Sp43及Sp452的N端胺基酸可能有修飾的現象而無法定序。在mRNA differential display實驗方面，經3種 anchor primer及12種 H-AP primer組合，可找出16個差異cDNA片段。以reverse Northern hybridization篩選出10個cDNA片段進行選殖。最後以Northern hybridization進行確認，結果有三個cDNA片段，它們各自代表的基因在老化階段表現增強，分別命名為pC20b、pG16-1及pA15e。pC20b為cathepsin B類蛋白質，進一步分析顯示應與Sp30-1及Sp30-2分屬不同subfamily。pG16-1與阿拉伯芥AtPK 2324 receptor-like serine/threonine protein kinase有很高的相似度。pA15e則在GenBank中比對不到明顯相似的基因。

Leaf senescence is the final stage of leaf development in higher plants. During this stage, senescing leaves recycle their movable nutrients to other parts of the plant. Nuclear genes are known to be involved in the senescence process, recent evidence indicates that leaf senescence is a form of programmed cell death (PCD). Molecular approaches have been recently employed to identify the senescence-associated genes (SAGs).In this thesis, two dimensional gel electrophoresis of proteins and mRNA differential display (DD-PCR) were used to study leaf senescence in sweet potato. By using two dimensional gel electrophoresis, four protein spots were identified to be leaf senescence-specific. They are named Sp30-1, Sp30-2, Sp43 and Sp45 according to the molecular mass estimated on the SDS-PAGE gel. After N-terminal sequencing and database comparison, Sp30-1 and Sp30-2 were found to be members of the cathepsin protein. The identities of Sp43 and Sp45 remain unknown, possibly due to N-terminal blocking. Utilizing DD-PCR method with 3 anchor primers and 12 H-AP primers, 16 differential-displayed cDNA fragments were obtained. These 16 cDNA fragments were first screened with reverse Northern hybridization, and 10 positive cDNA fragments were subjected to cloning. Three cDNA clones including pC20b, pG16-1 and pA15e were confirmed to be senescence-specific by Northern hybridization. pC20b was found to belong to cathepsin B subfamily. Further analysis showed that Sp30-1, Sp30-2 and pC20b are different cathepsin proteins. pG16-1 was found to be a receptor-like serine/threonine protein kinase. pA15e has no homologous gene sequence reported in the GenBank.

中文摘要 iAbstract iiList of abbreviation iii前言 1~13材料與方法 14~27結果 28~31討論 32~39參考文獻 40~50圖表 51~77附錄 78~79

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