臺灣博碩士論文加值系統

愛玉子 (Ficus awkeotasang Makino)為台灣特有種植物，用水搓洗愛玉種子所製得的愛玉凍為夏日常見的消暑飲品；其中也含有不少蛋白質，已鑑定出的蛋白質包括果膠甲基酯酶 (pectin methylesterase, PME)、幾丁質水解酵素 (chitinase)及似索馬甜蛋白(thaumatin-like protein, TLP)，和微量的病程相關之蛋白，如:第一類病程相關蛋白 (PR-1)及第四類病程相關蛋白 (PR-4)。目前已知的PR-4可以分為N端含有額外的幾丁質結合區域的Class I，以及缺乏此區域的Class II。而愛玉PR-4基因(FaPR4) 已經被選殖出來。演譯所得之蛋白質N端含有額外的幾丁質結合區域，其C端有一小段液泡序列 (vacuolar signal)。利用引子設計及PCR技術將液泡序列去除得到FaPR4(noCt) cDNA片段，並成功地在酵母菌Pichia分泌表達出r-FaPR4(noCt)及r-FaPR4(noCt/mutant)。r-FaPR4(noCt/mutant)為r-FaPR4(noCt)中一個胺基酸發生點突變。FaPR4(NO)為去除幾丁質結合區域的愛玉PR-4。 r-FaPR4、r-FaPR4(NO)和r-FaPR4(NO/mutant)亦已在酵母菌Pichia分泌表達出來。將純化後的r-FaPR4、r-FaPR4(noCt) 、r-FaPR4(noCt/mutant)、r-FaPR4(NO)和r-FaPR4(NO/mutant)進行RNase活性分析，發現以不同鹽濃度沖提出的蛋白質具有不同程度的RNase活性。而重組蛋白之抗真菌能力分析結果發現r-FaPR4、r-FaPR4(noCt) 及r-FaPR4(noCt/mutant)對Glomerella cingulata及Fusarium oxysporum的孢子萌芽具有抑制活性，且重組蛋白經加熱後還具有一定的活性，其中以r-FaPR4的抗真菌活性最好。進一步研究結果顯示這些重組蛋白會使Rhizoctonia solani菌絲產生ROS反應 (reactive oxygen species)，其中以r-FaPR4所造成的ROS反應最明顯。

Jelly fig (Ficus awkeotasang Makino) is one of the endemic plants in Taiwan. The water extract from jelly fig achenes forms jelly curd, a common summer beverage. Some pericarpial proteins extracted from the jelly curd have been identified as a pectin methylesterase (PME), chitinase, thaumatin-like protein (TLP), two PR proteins, pathogenesis related-1(PR-1) and pathogenesis related-4 (PR-4). PR-4 was classified into two groups, Class I contains extra chitin binding domain (CBD) in N-ternimus and Class II has no such domain. cDNA fragment coding for Jelly fig PR4 (FaPR4) has been cloned and its deduced protein has CBD in N-terminus and a vacuolar signal in C-terminus. Using designed primers and PCR technology obtained cDNA fragnmemt encoding FaPR4(noCt), a jelly fig PR-4 without vacuolar signal. FaPR4(noCt) and FaPR4(noCt/mutant) were successfully expressed in yeast Pichia pastoris. FaPR4(noCt/mutant) is a point-mutant of FaPR4(noCt). The purified r-FaPR4, r-FaPR4(noCt), r-FaPR4(noCt/mutant), r-FaPR4(NO) and r-FaPR4(NO/mutant) were subjected for RNase activity assay. The recombinant proteins eluted from different salt concentrate showed different RNase activity. Recombinantion r-FaPR, r-FaPR4(noCt)and r-FaPR4(noCt/mutant) exhibited antifungal activity toward Glomerella cingulata and Fusarium oxysporum. After heating at 100oC for 10 min, the recombination proteins remained some activity, and r-FaPR4 had the highest antifungal activity. In addition, recombination proteins could cause ROS response in Rhizoctonia solani hyphae, and r-FaPR4 has the most obvious effect on ROS response.

目錄
摘要........................................................i
Abstract...................................................ii
誌謝......................................................iii
表目錄....................................................vii
圖目錄...................................................viii
第一章 前言.................................................1
第二章 文獻回顧.............................................2
2.1 愛玉瘦果及其已知水溶性蛋白簡介..........................2
2.2 植物防禦系統與PR蛋白簡介................................3
2.3 PR-4 protein簡介......................................4
2.4 酵母菌Pichia pastoris表達系統...........................6
第三章 實驗材料與方法.......................................9
3.1 實驗材料................................................9
3.2 聚合酶鏈鎖反應 (Polymerase chain reaction,PCR).........9
3.2.1 引子設計..............................................9
3.2.2 聚合酶鏈鎖反應 (PCR)..................................9
3.2.3 PCR產物純化 (PCR clean up)............................9
3.3 質體DNA之純化 (Purification of plasmid DNA).............9
3.4 限制酶反應 (Restriction enzymes digestion).............10
3.5 DNA膠體純化 (DNA gel extraction)......................10
3.6 接合反應 (Ligation)....................................10
3.7 轉形作用-熱休克 (Heat Shock)...........................11
3.7.1 勝任細胞之製備.......................................11
3.7.2 熱休克細胞轉形.......................................11
3.7.3 重組質體之篩選確認...................................11
3.8 以酵母菌為宿主進行蛋白表現.............................11
3.8.1 P. pastoris X-33 勝任細胞之製備......................11
3.8.2 線性重組質體pGAPZαA-PR4(noCt)之製備.................12
3.8.3 P.pastoris X-33之轉形................................12
3.8.4 pGAPZαA-PR4(noCt)重組菌株之篩選.....................12
3.8.5 以酵母菌為宿主之蛋白大量表達.........................12
3.8.6 以陰離子管柱 (HiTrapTM Q FF)純化重組蛋白.............12
3.8.7 西方墨點法 (Western blot)............................13
3.9 RNase活性測試..........................................13
3.9.1 RNA之抽取............................................13
3.9.2 電泳分析蛋白質的RNase活性............................13
3.10 抗真菌活性測試........................................14
3.10.1 抑制真菌孢子萌芽測試................................14
3.10.2 抑制Sclerotium rolfsii菌核之菌絲生長測試............14
3.10.3 ROS (reactive oxygen species) response觀察.........14
第四章 結果...............................................15
4.1 愛玉瘦果第四類病程相關蛋白FaPR4(noCt)及FaPR4(noCt/mutant)之基因重組.................................................15
4.1.1 FaPR4(noCt)及FaPR4(noCt/mutant)基因之PCR片段純化.....15
4.1.2 pGAPZαA-PR4(noCt)及pGAPZαA-PR4(noCt/mutant)重組質體之構築.......................................................15
4.1.3 轉形後重組質體之確認.................................15
4.1.3.1 PCR確認............................................15
4.1.3.2 限制酶確認.........................................15
4.2 以酵母菌Pichia pastoris為宿主進行重組蛋白質之分泌表達及純化.........................................................16
4.2.1 r-FaPR4(noCt)重組蛋白之酵母菌分泌表達................16
4.2.2 r-FaPR4(noCt/mutant)重組蛋白之酵母菌分泌表達.........16
4.2.3 以陰離子管柱純化酵母菌分泌表達之r-FaPR4蛋白..........16
4.2.4 以陰離子管柱純化酵母菌分泌表達之r-FaPR4(noCt)蛋白....16
4.2.5以陰離子管柱純化酵母菌分泌表達之r-FaPR4(noCt/mutant)蛋白.........................................................17
4.2.6 以陰離子管柱純化酵母菌分泌表達之r-FaPR4(NO)蛋白......17
4.2.7以陰離子管柱純化酵母菌分泌表達之r-FaPR4(NO/mutant)蛋白
...........................................................17
4.3 以西方墨點法辨識PR-4之重組蛋白.........................17
4.4 重組蛋白之RNase活性測試................................18
4.4.1 陰離子管柱純化buffer及重組蛋白r-FaPR4之RNase活性測試.........................................................18
4.4.2 重組蛋白r-FaPR4(noCt)及r-FaPR4(noCt/mutant)之RNase活性測試.......................................................18
4.4.3 重組蛋白r-FaPR4(NO)及r-FaPR4(NO/mutant)之RNase活性測試.........................................................18
4.4.4 重組蛋白PR1-1、X-33及buffer過管柱後之RNase活性測試.........................................................18
4.4.5測試是否在蛋白質樣品中有抑制RNase活性之物質及鹽濃度對於重組蛋白r-FaPR4的 RNase活性影響............................19
4.4.6測試鹽濃度對於重組蛋白r-FaPR4(noCt) 的RNase活性影響.........................................................19
4.4.7測試使用於抗菌活性之重組蛋白是否具有RNase活性及加熱後的影響.......................................................20
4.5 抗真菌活性測試.........................................20
4.5.1 抑制真菌G.cingulata孢子萌芽測試......................20
4.5.2 抑制真菌F. oxysporum孢子萌芽測試.....................21
4.5.3 抑制S. rolfsii菌核之菌絲生長測試.....................21
4.5.4 ROS response觀察....................................22
第五章 討論...............................................23
第六章 結論...............................................26
參考文獻...................................................60
附錄.......................................................64
Extended Abstract..........................................66
簡歷(CV)...................................................72

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