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研究生:蘇正庭
研究生(外文):Su Cheng-Ting
論文名稱:甘藷葉片老化相關基因半胱胺酸蛋白分解酶SPCP2於轉殖阿拉伯芥之研究
論文名稱(外文):studies of a sweet potato leaf senescence-associated cysteine protease SPCP2 gene in transgenic Arabidopsis plants
指導教授:陳顯榮陳顯榮引用關係
指導教授(外文):Chen Hsien-Jung
學位類別:碩士
校院名稱:中國文化大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2005
畢業學年度:94
語文別:中文
論文頁數:70
中文關鍵詞:老化半胱胺酸蛋白分解酶儲存性蛋白質
外文關鍵詞:senescencecysteine proteasesstorage protein
相關次數:
  • 被引用被引用:2
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  • 下載下載:28
  • 收藏至我的研究室書目清單書目收藏:1
最近的研究顯示種子液泡中的儲藏性蛋白質,於種子成熟期間的累積或發芽過程被降解以作為幼苗養分的來源,這些過程皆與一群半胱胺酸分解酵素(cysteine proteases)有關;包括有legumain-like 的asparaginyl endopeptidases (或稱為vacuolar processing enzymes) 及其下游受調控的papain-like 的cysteine protease。本實驗室於甘藷葉片老化過程中也發現相類似的legumain-like asparaginyl endopeptidase SPAE及papain-like 的cysteine protease (SPCP2),因此推論甘藷葉片老化過程似乎使用與種子發芽相類似的機制以進行蛋白質的降解與再利用。本實驗將已分離出的SPCP2之全長cDNA先構築於pBI121的載體中,再利用農桿菌轉殖技術以floret dip method送入阿拉伯芥植株中,利用抗抗生素Kanamycin之特性篩選出17株轉殖T0阿拉伯芥植株,並以genomic PCR及RT-PCR的方法來證實SPCP2於轉殖植株中確實存在並且有表現。由外觀型態分析顯示,轉殖T1植株比對照組植株有較早開花之現象、果莢發育不完全率較多、T1與T2種子的鮮重較輕與發芽率較差之情形。基於這些研究的結果,認為甘藷葉片老化可能使用與種子發芽過程儲存性蛋白質globulin(鹽溶性蛋白質)被降解之相類似機制,來進行蛋白質的降解及運移作用,而且SPCP2可能會促使植株提早由營養生長階段進入生殖生長階段。
Recent studies show that seed storage protein deposit during maturation or degradation/mobilization during germination are likely associated with a novel group of cysteine proteases such as legumain-like asparaginyl endopeptidase (or named as vacuolar processing enzyme) and papaine-like cysteine protease. In our laboratory, similar legumain-like asparaginyl endopeptidase, SPAE, and papaine-like cysteine protease, SPCP2, have been isolated from sweet potato senescent leaves. Therefore, a putative role of SPAE and SPCP2 in sweet potato senescent leaves for bulk protein degradation and mobilization similar to the mechanism utilized in seed for storage protein degradation/mobilization during germination. In this research, the isolated, full-length cDNA of SPCP2 was constructed in recombinant pBI121 vector and transferred into Arabidopisis with Agrobacteria tumefaciens and floret dip transformation method. About 17 transgenic Arabidopsis plants were isolated that showed resistance to Kanamycin. Genomic PCR and RT-PCR analysis also demonstrated the existence and expression of SPCP2 in transgenic Arabidopsis plants. Morphological analysis showed that transgenic T1 plants exhibited higher incomplete capsule development percentage, lighter Fw and lower germination percentage of T1 and T2 seeds, and earlier flowering when compared to control plants. Based on these results, we conclude that a mechanism utilized for seed storage protein degradation/mobilization during germination may also be adopted by sweet potato senescent leaves, and senescent-associated gene, SPCP2, may slightly promote early flowering from vegetative phase transition to reproductive phase.
目 錄
頁次
目錄 ……………………………………… Ⅰ
內容目次 ………………………………… Ⅱ
圖目錄 …………………………………… VI
表目錄 ……………………………………VIII
中文摘要 ………………………….……… IX
英文摘要 ……………………………….… X
附錄一 ……………………………….…… XI
附錄二 ……………………………………. XII
附錄三 ……………………………………. XIII








內容目次
頁次
壹、前言:
一、序言 ……………………………………………………………1.
二、葉片老化的重要性 ……………………………………………1.
三、葉片的老化 ……………………………………………………2.
四、種子的構造與發育 ……………………………………………7.
五、C/N Ratio對於植物生理之影響 ……………………………...9.
六、前人研究 ……………………………………………………...10.
貳、材料與方法:
I、實驗材料:
(一)甘藷 ……………………………………………………….13.
(二)甘藷 Cysteine protease SPCP2 cDNA ……………….…...13.
II、實驗方法:
一、阿拉伯芥轉殖植株:
(一)重組載體的構築:
A. 重組載體pGS轉型(transformation)
至E.coli DH5α勝任細胞 ..…………………………...14.
B.重組載體pGS質體DNA的分離 ……………………..15.
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C.利用PCR擴增cDNA片段 ….………………………...15.
D. 膠體中DNA之回收(gel extraction)..........................17.
E. 黏合反應(ligation)…………………………………...17.
F. 限制酶切割 ……………………………………………..18.
(二)、阿拉伯芥轉殖:
A.農桿菌勝任細胞的製備與電穿孔轉殖 ………………...19.
B.轉型農桿菌的檢測 ……………………………………...20.
C. floret dip transformation method花序轉型實驗 ..……...21.
D. 轉殖阿拉伯芥T0植株的篩選 ………………………..22.
E. 轉殖阿拉伯芥T0植株之種植、開花及種子採收..…...22.
(三)、轉殖阿拉伯芥T1植株的定性分析:
A. T1種子鮮重與發芽率之測定 ………………………….23.
B. 轉殖阿拉伯芥T1植株X2-test之分析 ………………..23.
C. Genomic DNA之抽取與Genomic PCR ………………..24.
D. Total RNA之分離與RT-PCR …………………………...26.
E. 轉殖阿拉伯芥T1植株外觀型態之觀察 ........................28.
F. 轉殖阿拉伯芥T2種子鮮重與發芽率之測定 .................28.



頁次
二、融合蛋白質的大量表現:
(一)重組載體的構築與融合蛋白質之誘導: A. 重組載體pETY166之構築 ...........................................29.
B. 融合蛋白質之誘導 …………………………………….30.
(二)SDS-PAGE電泳分析:
A. 藥品配製 ……………………………………………….30.
B. 膠體濃度表 …………………………………………….32.
參、結果:
Ⅰ、結果:
一、老化相關基因半胱胺酸蛋白分解酶基因重組載體之構築 ...33.
二、農桿菌之基因轉殖 ……………………………………………34.
三、轉殖阿拉伯芥T0植株之篩選 ………………………….……35.
四、轉殖阿拉伯芥T1植株之定性分析 …………………………..35.
五、轉殖阿拉伯芥T1植株外觀型態之比較 ……………………..38.
六、融合蛋白質的大量表現 ………………………………………39.
肆、討論:
一、轉殖阿拉伯芥植株之轉殖效率 ………………………………41.
二、轉殖阿拉伯芥植株含有SPCP2基因…………………………..41.
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三、SPCP2表現會促進轉殖T1植株提早開花…...………………42.
四、轉殖T1植株花序中SPCP2表現會影響種子與果莢發育………………………………………………………………...43.
五、種子鮮重與發芽率的影響……..……………………………...44.

參考文獻 ………………………………………………………...........47.



























圖目錄
頁次
圖一、老化相關基因半胱胺酸蛋白分解酶(SPCP2)之DNA及氨
基酸序列……………………………………………………….52.
圖二、含SPCP2重組pGEM-T easy vector之構築…………………53.
圖三、含SPCP2基因之重組pBI121載體之構築…………………..54.
圖四、含SPCP2基因之不同重組pBI121載體其方向性之檢測.…..55.
圖五、轉型農桿菌含重組pBI121載體之鑑定..……………………..56.
圖六、轉型農桿菌的檢測 ……………………………………………57.
圖七、阿拉伯芥T0轉殖植株之篩選…………………………………58.
圖八、轉殖T1阿拉伯芥植株利用genomic PCR檢測SPCP2基
因的存在與否…………………..…………………………...…59.
圖九、轉殖T1阿拉伯芥植株利用RT-PCR鑑定SPCP2之基因表
現 ………….…………………………………………………..60.
圖十、阿拉伯芥果莢及種子外觀型態分析………………..………...61.
圖十一、不同類型果莢內的種子數目………………………………..62.
圖十二、對照組及轉殖T1阿拉伯芥植株果莢發育不完全率之比
較……………………………………………………………….63.
圖十三、對照組及轉殖T1阿拉伯芥植株開花性狀之比較I…….....64.
圖十四、對照組及轉殖T1阿拉伯芥植株開花性狀之比較II…...….65.
圖十五、直接播種生長於土中之對照組及轉殖T1植株利用genomic
PCR進行SPCP2基因之檢測 ………………………….…….66.
圖十六、含SPCP2基因之重組載體pETY166之PCR檢測…….…67.
圖十七、含pETY166重組載體之E.coli(BL21(DE3))誘導SPCP2融合蛋白質之大量表現……………………………………..….68.












表目錄
頁次
表一、發芽於含有Kanamycin培養基之T1種子,記錄其外觀
型態(綠色或白色)以Chi-square test分析,推算T0轉
殖植株所含SPCP2之套數 ..…………………………..……..69.
表二、轉殖阿拉伯芥植株(T0與T1)產生的後代種子(T1與
T2)之鮮重及發芽率分析 …………………………………...70.
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