植物半胱胺酸蛋白酶抑制劑 ( phytocystatin ) 其作用為抑制半胱胺酸蛋白酶 ( cysteine proteases ) 的活性，主要分為三大群: 第一群 ( 約12-16 kDa )、第二群 ( 約23 kDa ) 及第三群( 約80 kDa )，於植物生長發育過程或對抗環境逆境方面扮演不同的生理功能及角色。本研究利用扣減雜交 ( subtractive hybridization ) 及RACE PCR從甘藷的老化葉片選殖到一段全長cDNA，命名為 SPCPI，其開放閱讀框架 ( open reading frame; ORF ) 含有765個核苷酸 ( 254個胺基酸 )，與第二群植物半胱胺酸蛋白酶抑制劑胺的基酸序列包括千穗谷、甘藍、番茄、矮牽牛、芝麻、馬鈴薯等具有高相似性 ( 約57至69% )，基因及蛋白質結構分析顯示其含有植物半胱胺酸蛋白酶抑制劑特有的保守胺基酸序列 ”LARFAVEEHNK”，可能參與抑制papain-like ( GG、QVVAG、PW )及抑制legumain-like ( SNSL ) 半胱胺酸蛋白酶的活性位點。甘藷半胱胺酸蛋白酶抑制劑SPCPI於發芽塊根、L3成熟及L4老化中期葉片中皆有顯著表現，且受ethephon及NaCl處理誘導。Ethephon 及NaCl處理顯著促進葉片老化、H2O2以及MDA含量增加，外加甘藷半胱胺酸蛋白酶抑制劑SPCPI融合蛋白質可延緩ethephon誘導的葉片老化，然而卻加速NaCl處理誘導的葉片老化。前處理包括95℃加熱5分鐘或室溫下先混合anti-SPCPI多株抗體兩小時，可以顯著抑制外加的甘藷半胱胺酸蛋白酶抑制劑SPCPI融合蛋白質的作用，回復至原來ethephon或NaCl處理誘導的葉片老化、H2O2以及MDA含量增加的程度。另外單獨外加甘藷半胱胺酸蛋白酶抑制劑SPCPI融合蛋白質並無顯著加速黑暗處理甘藷葉片老化、H2O2以及MDA含量增加的速率。根據上述實驗結果結論甘藷第二群半胱胺酸蛋白酶抑制劑SPCPI為ethephon 及NaCl可誘導的基因，其生理功能可能與調整ethephon 及NaCl誘導的甘藷葉片老化、H2O2以及MDA含量增加有關，且需要ethephon 及NaCl可誘導的交互作用因子一起協同作用。

Plant phytocystatins are cysteine protease inhibitors and generally classified into three groups: group I ( ca. 12-16 kDa ), group 2 ( ca. 23 kDa ) and group 3 ( ca. 80 kDa ). Phytocystatins play diverse physiological roles and function in cope with plant developmental cues and environmental stress responses. In this research, a full-length cDNA, SPCPI, has been cloned from sweet potato senescent leaves with PCR-based subtractive hybridization and RACE PCR. Sweet potato SPCPI contains 765 nucleotides ( 254 amino acids ) in its open reading frame ( ORF ), and exhibits high amino acid sequence identities ( ca. 57% to 69% ) with different plant phytocystatins, including Amaranthus hypochondriacus, Brassica oleraceae, Solanum lycopersicum, Petunia hybrid, Sesamum indicum, and Solanum tuberosum. Gene and protein structural analysis showed that the SPCPI-encoded protein contains the unique conserved amino acid sequence motif “LARFAVEEHNK” of phytocystatin, potentially involved in inhibiting active sites of papain-like ( GG, QVVAG, PW ) and legumain-like ( SNSL ) cysteine proteases. Gene expression patterns showed that phytocystatin SPCPI was significantly enhanced in sprouting storage roots, L3 mature and L4 early senescent leaves, and highly inducible by ethephon, an ethylene-releasing compound, and NaCl. Ethephon and NaCl treatment significantly promoted leaf senescence, H2O2 and MDA elevation. Exogenous purified phytocystatin SPCPI fusion protein significantly mitigated ethephon-mediated leaf senescence, however, remarkably accelerated NaCl-induced leaf senescence. The SPCPI fusion protein-mediated effects could be remarkably antagonized by pre-treatment with anti-SPCPI antibody at room temperature for 2 h or by boiling at 95℃ for 5 min before its application, and reversed them to similar levels of leaf senescence, H2O2 and MDA elevation caused by ethephon or NaCl alone. In addition, exogenous phytocystatin SPCPI fusion protein alone did not significantly alter dark-induced leaf senescence, H2O2 and MDA elevation. These results conclude that sweet potato group II phytocystatin SPCPI is an ethephon and NaCl-inducible gene, and likely participates in the modulation of ethephon and NaCl-mediated leaf senescence, H2O2 and MDA elevation, which require ethephon or NaCl-inducible interacting components for effective interactions.

目 錄
論文審定書…………………….………………………………………………………….......i
誌謝…………………….…………………………………………………………................ii
中文摘要…………………….………………………………………………………….........iii
英文摘要…………….………………………………………………………………............iv
目錄…………………………………………………………………….…………………...…v
圖次................................................................................................................viii
壹、緒論……………………………………………………………………………............1
甘藷……………….……………………………………………………………………….1
葉片老化的生理過程及機轉……………..…………………………………................1
影響葉片老化的因子…..……………………………………………..........................2
乙烯與葉片老化……………………………..………………………..........................3
鹽分逆境與葉片老化………………………………………………….........................4
老化相關基因半胱胺酸蛋白酶 ( cysteine protease ) ………................................5
植物半胱胺酸蛋白酶抑制劑 ( phytocystatin ) 的蛋白質結構分析……….…...........6
植物半胱胺酸蛋白酶抑制劑 ( phytocystatin ) 在生長發育過程或環境逆境
下扮演的功能及角色......................................................................................8
研究動機與目的...........................................................................................11
貳、實驗材料..................................................................................................12
甘藷...........................................................................................................12
分析基因表現的引子....................................................................................12
參、實驗方法..................................................................................................13
甘藷半胱胺酸蛋白酶抑制劑 ( cysteine protease inhibitor ) SPCPI之基因選殖
及定性分析.................................................................................................13
甘藷半胱胺酸蛋白酶抑制劑SPCPI基因選殖及生物資訊學分析..........................13
甘藷半胱胺酸蛋白酶抑制劑SPCPI生長發育表現分析.......................................13
甘藷半胱胺酸蛋白酶抑制劑SPCPI組織專一性表現分析....................................13
甘藷半胱胺酸蛋白酶抑制劑SPCPI塊根不同發育時期表現分析..........................14
SPCPI重組表現載體構築與誘導融合蛋白質表現、純化及抗體製備...................14
甘藷成熟葉片處理.......................................................................................15
不同濃度ethephon處理甘藷成熟葉片.............................................................15
不同濃度NaCl處理甘藷成熟葉片....................................................................15
甘藷半胱胺酸蛋白酶抑制劑SPCPI 融合蛋白質處理甘藷成熟葉片......................16
外加SPCPI 融合蛋白質處理甘藷成熟葉片......................................................16
外加不同劑量SPCPI 融合蛋白質及ethephon處理甘藷成熟葉片.........................16
外加SPCPI融合蛋白、anti-SPCPI抗體及Ethephon處理甘藷成熟葉片…….……..17
外加失活之SPCPI 融合蛋白質及ethephon處理甘藷成熟葉片……….…………….17
外加不同劑量SPCPI 融合蛋白質及NaCl處理甘藷成熟葉片...............................17
外加SPCPI融合蛋白、anti-SPCPI抗體及NaCl處理甘藷成熟葉片…………....…..18
外加失活之SPCPI 融合蛋白質及NaCl處理甘藷成熟葉片……….………………....18
扣減雜交法……………………………………………………………………………...19
Rapid amplification of cDNA ends……...........................................................27
融合蛋白質SPCPI的純化..............................................................................30
葉綠素含量測量...........................................................................................31
光合作用效率測量 ( Fv/Fm ) .........................................................................31
DAB 染色分析.............................................................................................32
H2O2 含量分析...........................................................................................32
MDA含量分析..............................................................................................32
SPCPI基因表現的分析 ( RT-PCR& Western blot ) ..........................................33
Total RNA 抽取............................................................................................33
RT-PCR......................................................................................................34
PCR...........................................................................................................35
Western blot...............................................................................................37
肆、結果.........................................................................................................40
甘藷半胱胺酸蛋白酶抑制劑SPCPI的生物資訊學分析.......................................40
甘藷半胱胺酸蛋白酶抑制劑SPCPI在不同葉片生長發育階段及組織專一性
的表現........................................................................................................40
甘藷半胱胺酸蛋白酶抑制劑SPCPI在不同塊根發芽週數的表現..........................41
甘藷半胱胺酸蛋白酶抑制劑 SPCPI 基因表現受 ethephon 誘導.........................42
甘藷半胱胺酸蛋白酶抑制劑 SPCPI 基因表現受 NaCl 逆境誘導.........................43
甘藷半胱胺酸蛋白酶抑制劑 SPCPI 融合蛋白的誘導與純化...............................45
甘藷半胱胺酸蛋白酶抑制劑 SPCPI 融合蛋白延緩 ethephon 誘導的甘藷葉片老
化..............................................................................................................45
前處理 anti-SPCPI 抗體或熱處理 SPCPI 融合蛋白對 ethephon誘導甘藷老
片老化的影響...............................................................................................46
外加甘藷半胱胺酸蛋白酶抑制劑 SPCPI 融合蛋白促進 NaCl 誘導的甘藷葉片老
化...............................................................................................................49
前處理 anti-SPCPI 抗體或熱處理抑制 SPCPI 融合蛋白對 NaCl 誘導甘藷葉片老化
的影響........................................................................................................50
單獨外加甘藷半胱胺酸蛋白酶抑制劑 SPCPI 融合蛋白沒有顯著增加黑暗處理下甘
藷葉片老化速率...........................................................................................53
伍、討論.........................................................................................................55
陸、參考文獻..................................................................................................59

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