臺灣博碩士論文加值系統

食肉植物大多生長在土壤貧瘠的地區，並且各自演化出不同的捕食器官以便從其他生物身上獵取其所缺乏的氮源及磷源。現今對食肉植物的研究主要集中在他們的演化、形態以及少數物種的蛋白質體分析，鮮少有人在基因與轉殖上多作著墨。谷霍腺毛草 (Byblis guehoi) 又稱為彩虹草，有著生長快速、構造簡單且佈滿大量分泌型腺毛的特性，所以我們選擇用它做為研究對象。經由共軛交顯微鏡分析發現彩虹草的葉子上共有三種腺毛，長型、短型與基底型。以組織貼盤測試可知基底型腺毛可分泌蛋白酶與磷酸酶，然而長型與短型腺毛則無明顯上述活性。經嘗試多種萃取與純化方式後，我們成功從極具黏性的葉子表面收集到蛋白，膠體電泳分析得知蛋白主要集中於 72 kDa 的位置，將之回收進行蛋白質質譜身分鑑定後，發現其中至少有五種可能的蛋白質，包括蛋白酶與磷酸酶等，進一步使用陰離子交換樹脂 (Q-column)分離蛋白質，發現蛋白酶與磷酸酶這兩種主要蛋白的電性差異極大。為了找出主要蛋白的基因序列，將質譜比對到的物種蛋白進行比對分析後，設計退化引子 (degenerate primer)，並以彩虹草的 cDNA 為模板 進行PCR 放大後，已得到部分的磷酸酶序列片段，且並非NCBI網站已有的序列片段。未來將進行 RACE 找出目標蛋白的基因序列全長，再向上游搜尋相應的啟動子序列 (secretion signal peptide)。而在轉殖系統的建構上，初步發現彩虹草可以透過農桿菌的感染暫時性表現 GUS 活性，暗示未來可使用農桿菌進行彩虹草的基因轉殖。由組織培養測試可知彩虹草對 kanamycin 有很高的抗性，然而對 hygromycin 卻非常的敏感。後續的實驗將使用分泌型綠色螢光蛋白表現載體，對彩虹草的外泌系統進行評估與測試。本實驗的最終目標是使用彩虹草生產外源蛋白，尤其是需要被外泌的蛋白，並達到只需洗過就能回收的效果。

Carnivorous plants usually grow in nutrient-poor wetlands. They have evolved specialized organs to capture and digest small organisms for nutrition. Researches today for carnivorous plants are mostly focused on their evolution, morphology, classification, with some proteomic studies but rarely at the gene levels. Here we used Byblis for further research due to its rapid growth, simple structure and very large amount of dew drop secretion. Three kinds of the trichomes, long, short and basal forms, were observed under confocal microscope. Tissue printing on substrate plate revealed activities of protease and phosphatase, likely secreted from the basal forms but not long or short forms of the trichome. To recover proteins from the viscous dew drop, several extraction methods were tested. One major banding at about 72 kDa was detected on SDS-PAGE. LC-MS/MS analysis reveals five candidate proteins including one subtilisin-like protease and one purple acid phosphatase. These two proteins showed very different affinities in the ion-exchanger (Q column) chromatography, suggested their differences in isoelectric points of proteins. We performed degenerate PCR on Byblis cDNA and obtained one DNA fragment that putatively encodes purple acid phosphatase. Further RACE and genomic PCR will be used to obtain the full-length cDNA and promoter fragments. Moreover, transient Agrobacterium infiltration to Byblis stem caused a prominent GUS signal, suggested that the Agrobacterium-mediated transformation may be feasible. Antibiotic resistance test showed that Byblis had a high tolerance to kanamycin but were very sensitive to hygromycin. Tissue culture and plant regeneration protocols will be tested for the purpose of Byblis transformation. Our final goal is to build up a Byblis bioreactor in which the engineered proteins can be secreted and harvested simply by wash.

摘要 i
Abstract ii
目錄 iii
壹、前言 1
一、食肉植物 1
二、植物的腺毛 (glandular trichome) 1
三、蛋白的生物反應器 (bioreactor) 2
四、研究動機及目的 3
貳、材料與方法 4
參、結果 12
一、彩虹草的形態分析 12
(一) 以解剖顯微鏡觀察葉表面結構 12
(二) 以共軛焦顯微鏡觀察彩虹草腺毛 12
二、彩虹草表面分泌蛋白萃取 12
(一) 彩虹草表面黏液中蛋白萃取方法之建立 12
(二) 彩虹草表面分泌蛋白之鑑定與活性分析 13
(三) 以電性分離彩虹草表面分泌蛋白與分析 13
三、彩虹草磷酸酶與蛋白酶基因序列的選殖 14
(一) 彩虹草 PAP 未知序列的選殖 14
(二) 彩虹草 SBT 未知序列的選殖 15
四、彩虹草轉殖系統之建立 15
(一) 彩虹草轉殖條件測試 15
(二) 分泌型蛋白載體構築 16
肆、討論 18
伍、參考文獻 24
陸、參考網址 27
柒、圖表 28
表一、質譜鑑定資料校正後的五個候選蛋白與可能功能 28
表二、質譜鑑定資料中 PAP 所比對到的物種與胜肽序列 29
表三、質譜鑑定資料中 SBT 所比對到的物種與胜肽序列 30
表四、本研究所使用之核酸引子序列 31
圖一、彩虹草實驗流程圖 32
圖二、彩虹草葉子表面分泌型腺毛分析 33
圖三、以共軛焦顯微鏡對 PI 染色的彩虹草進行分析 34
圖四、以共軛焦顯微鏡對 calcoflour white 和 auramine O 染色的彩虹草進行分析 35
圖五、迷你真空吸引裝置示意圖與實體照 36
圖六、彩虹草總表面蛋白初萃取分析 37
圖七、以酪蛋白瓊脂盤測試彩虹草各部位蛋白酶活性 38
圖八、以BCIP瓊脂盤測試彩虹草葉磷酸酶活性 39
圖九、以 Q-column 分離彩虹草表面初萃蛋白與丙酮沉澱 40
圖十、以銀染分析彩虹草各鹽濃度流洗液 41
圖十一、各陰離子交換樹脂分離液之蛋白酶及磷酸酶活性分析 42
圖十二、各物種間 PAP 序列比對 43
圖十三、 PAP 比對後引子設計區域與各物種間保留性示意圖 44
圖十四、 彩虹草 PAP 基因序列之選殖 45
圖十五、彩虹草 PAP 定序結果比對 46
圖十六、各物種間 SBT 序列比對 47
圖十七、GUS 在暫時性轉殖的彩虹草莖中的表現 48
圖十八、kanamycin 對彩虹草的毒性試驗 49
圖十九、hygromycin 對彩虹草的毒性試驗 50
圖二十、 彩虹草癒傷組織生長測試 51
圖二十一、以三輪 PCR 將訊號胜肽掛載至 mGFP DNA 序列上 52
圖二十二、將SP-GFP 以 pGEM®-T Easy 放大後切膠回收並接入 pEpyon-32H 53
圖二十三、p32H-cluv3sp 載體圖 54
圖二十四、質譜比對到的蛋白在不同型態腺毛作用的假說示意圖 55
圖二十五、彩虹草酵素分解網假說圖 56
附表一、植物的毛狀體類型 57
附圖一、2016 年度植物 APG IV 分類 (網址一) 58
附圖二、彩虹草形態特徵 59
附圖三、彩虹草表面蛋白萃取流程 (一) 60
附圖四、質譜比對蛋白與分子量及等電點評估分析 61
附圖五、彩虹草表面蛋白萃取流程 (二) 62
附圖六、pGEM®-T Easy 載體示意圖 63
附圖七、pCAMBIA 1305.1 載體示意圖 64
附圖八、訊號胜肽掛載示意圖 65
附圖九、以 coomassie blue 染色分析未加 DTT 處理的各鹽濃度流洗液 66
附圖十 (一)、現階段主要的植物反應器類型 67
附圖十 (二)、現階段主要的植物反應器類型 68

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