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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 樓國隆(Kuo-Long Lou) | |
dc.contributor.author | Ying-Chiu Tsai | en |
dc.contributor.author | 蔡贏萩 | zh_TW |
dc.date.accessioned | 2021-06-15T05:45:40Z | - |
dc.date.available | 2015-09-09 | |
dc.date.copyright | 2010-09-09 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-19 | |
dc.identifier.citation | Broekaert WF, Terras FR, Cammue BP, Osborn RW (1995) Plant defensins: novel antimicrobial peptides as components of the host defense system. Plant Physiol
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J Recept Res 14(6-8): 423-445 Yang YF, Cheng KC, Tsai PH, Liu CC, Lee TR, Lyu PC (2009) Alanine substitutions of noncysteine residues in the cysteine-stabilized alphabeta motif. Protein Sci 18(7): 1498-1506 David RH and James MC (1998) Pichia protocols 李信興 (Li Shin-Shing) (2002) 綠豆防禦素VrCRP 的突變與活性分析 國立海 洋大學水產生物科技研究所碩士論文 官振群 (Kuan Ching-Chun) (1998) 綠豆抗豆象相關cDNA 的表現與生物活性研 究 國立台灣大學農業化學研究所碩士論文 陳計志 (Chen Ji-Jr) (2003) 綠豆防禦素VrCRP 在Pichia pastoris 的表現與 功能分析 國立海洋大學生物科技研究所碩士論文 陳冠仲 (Chen Kuan-Chung) (2002) 綠豆抗豆象基因VrCRP 及VrArc 的表現與功能分析 國立台灣大學農業化學研究所博士論文 陳建宏 (Chen Gan-Hong) (2005) 乾豇豆屬植物防禦素基因之功能研究 國立台灣大學微生物與生化學研究所博士論文 陳詠哲 (Chen Yung-Che) (2001) 綠豆Vigna radiata VC 6089A 中抗豆象蛋白質 的純化及其活性分析 國立台灣大學農業化學研究所碩士論文 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47030 | - |
dc.description.abstract | 綠豆防禦素VrD1 (Vigna radiata defensin 1) 是一種由46個胺基酸所組成的植物防禦素,純化自綠豆品系VC 6089A的種皮,且經由種子餵食實驗發現其對綠豆豆象 (bruchids; Callosobruchus chinensis or C. maculatus) 具有毒殺的效果。VrD1為首度被發現除了抗真菌、細菌的活性之外,同時兼具抗昆蟲活性的植物防禦素; 然而其造成綠豆豆象死亡之活性來源及作用機制目前並不完全清楚。
本篇論文可分為兩部分: (I) 利用嗜甲醇酵母菌表現系統進行最適化綠豆防禦素VrD1突變株之製備: 本實驗室過去經由序列及結構比對,分別分析了VrD1與蠍毒蛋白 (scorpion toxins)、及多種具有抗菌活性植物防禦素的異同後,已經合理推斷出VrD1造成綠豆豆象死亡之活性來源應在於其分子一側與蠍毒具高度保留性之數個鹼性胺基酸。而分子另一側之數個鹼性胺基酸,則在與多種具抗菌活性的植物防禦素比較時具高度的序列保留性,推測應該與抗菌活性有關。 為了進一步探討這兩群序列高度保留性的鹼性胺基酸在VrD1抗蟲、抗菌活性上所扮演的角色,本實驗室過去製備了多株VrD1突變株,並進一步以嗜甲醇酵母 (Pichia pastoris) 表現、純化出數種VrD1的突變蛋白。然而後來經過定序檢測後,卻發現多數突變株有序列移碼 (frame-shift) 的問題;另一方面,這些突變株也普遍存在著蛋白表現不佳的情形,故而過去的電生理活性測定實驗會有樣本數不足的問題。因此本篇論文第一部份的主要方向有二:(1) 重新製備序列正確的突變株。(2) 重新挑出能夠表現高量VrD1蛋白的表現株,期望能改善過去產量不佳的狀況。 在本研究中,我們除了利用原本G418抗性篩選的方法來做為高重複基因片段 (high gene copy numbers) 及高表現量的指標之外,還另外加入了小量表現配合西方墨點法的分析方法,希望藉此能夠一次篩選多株表現株並直接看出它們表現能力的優劣; 也同時一方面能夠排除偽陽性抗G418活性的菌株,另一方面能從中找出具有最佳表現潛力的表現株。這些策略上的改變使得我們改善了過去大量表現後產量不佳的狀況。將R26A、K24A 之VrD1-pPIC9K質體重新送入嗜甲醇酵母菌後,自G418/YPD培養基選出七株菌落生長較佳的表現株,進一步利用小量表現、配合西方墨點法分析的篩選方法,我們篩出了Y43、Y38這兩株能成功地表現R26A VrD1的表現株; 西方墨點法的結果顯示Y38的訊號遠比Y43來的強。進一步比較它們大量表現後的產量,Y43表現量不佳,每公升產量只有300 μg;而Y38產量則比Y43高了15倍左右,每公升可產4.5 mg。大量表現的產量結果也與小量表現的分析結果趨勢相符。再者,目前利用定點突變法 (site-directed mutagenesis) 已成功將Wild type VrD1點突變為K7A VrD1。未來會繼續利用此法將剩餘的突變株製備出來。 本實驗的具體目標為落實產業化或產學合作。配合目前已建立的表現株篩選方法及定點突變法,我們將持續進一步設計多種VrD1突變序列,篩選出具有高抗蟲或抗菌活性的VrD1突變蛋白,期望能研發出具高抗蟲、抗菌活性、又可自然分解的生物性農藥。 (II) 尋找綠豆防禦素VrD1在昆蟲細胞膜上之標的蛋白及其抗蟲機制之探討: 根據過去的結構與序列比對結果,我們推論VrD1可能是使用類似蠍毒中的 short toxins 之相似機制與細胞膜上之鉀或氯離子通道作用;我們實驗室的電生理實驗結果也已初步排除了作用在氯離子通道的可能性。為了更深入地了解VrD1造成昆蟲細胞膜上大規模電流受抑制的分子機制,我們試圖以蛋白質體學的方法直接尋找VrD1作用在昆蟲細胞膜的標的蛋白。 依照一般研究蛋白交互作用 (protein-protein interaction) 方法學來說,實驗至少需要兩方面的材料: 【材料一】足量的VrD1蛋白: 我們一開始先嘗試以嗜甲醇酵母菌表現wild type VrD1,結果顯示雖然能成功獲得 VrD1 產物,然而產量並不理想。故接著我們改成直接由綠豆來取得天然的 VrD1 蛋白,結果成功地獲得了足量且純度高的VrD1產量,每500公克綠豆可以萃取出5~6 mg的VrD1蛋白,進一步以質譜儀鑑定也確定序列無誤。【材料二】受VrD1活性影響的昆蟲細胞株: 雖然之前全細胞電位鉗制量測法 (whole-cell voltage-clamp) 的實驗結果顯示VrD1能對秋行軍蟲細胞株 (Sf21, Sf9 cell lines) 的細胞膜電流產生大規模的抑制情形,然而這兩株細胞株卻會因為缺乏該物種 (秋行軍蟲) 的基因體資料庫,即使找到可能的候選標的蛋白也明顯地無法鑑定出其身分。其他目前已存在的昆蟲細胞株也幾乎存在這方面的問題。因此,我們認為已經建立完整基因體資料庫的果蠅細胞株 (Drosophila S2 cell line) 會是個不錯的選擇。在本論文中,我們以細胞存活率實驗來檢測VrD1是否會對果蠅細胞株存活產生影響,藉此判斷是否能以果蠅細胞株作為後續的實驗材料。 實驗結果發現果蠅細胞在經由飢餓處理 (starvation) 四小時後,在較低細胞密度、50 μM VrD1濃度的條件下處理72小時後,相較於未加藥組,加藥組的細胞存活數較少,在統計上有顯著差異性,細胞型態也可以觀察到明顯的細胞膜破裂現象。由以上結果我們合理推測VrD1對果蠅細胞有毒殺性。因此初步推論果蠅細胞株上很可能存在著會與VrD1相互作用的標的蛋白,有潛力作為我們尋找VrD1標的蛋白並進一步研究其活性機制的研究材料。果蠅是非常普遍的研究系統,相較於其他昆蟲細胞株在分生或細胞生物學的方法學上更有其便利性,果蠅細胞株會是一個深入研究VrD1分子機制的好實驗系統。我們未來會進一步以其他細胞生物學的方法研究細胞存活現象的機制,並進一步開始著手後續蛋白質體學的實驗。 | zh_TW |
dc.description.abstract | VrD1 (Vigna radiata defensin 1) is a member of the plant defensin family, containing 46 amino acids and four pairs of disulfide bonds. Isolation of a cDNA encoding a small cysteine-rich protein designated VrCRP (then also known as VrD1) from a bruchid-resistant mungbean revealed the first discovered plant defensin exhibiting both in vitro and in vivo insecticidal and antifungal activities. However, the molecular and structural basis of this unique insecticidal activity of VrD1 is still not fully understood.
This thesis is divided into two parts: I. Optimized preparation of VrD1 mutants expressed in Pichia pastoris: Based on the structural and sequence alignment, it is suggested that VrD1, in addition to γ-thionins and several amylase inhibitors, is highly homologous to scorpion toxins, especially the short toxins. We have deduced that VrD1 may utilize a newly found cluster of basic residues on one side of VrD1 molecule to achieve its insecticidal function, whereas another cluster of previously identified basic residues located on the other side of the molecule, which is conserved for all γ-thionins, should be used to achieve the antibacterial/antifugual activities for VrD1 and for all other plant defensins. In order to understand the roles of this newly found cluster of conserved basic residues, we have constructed several expression strains for VrD1 mutants and purified these mutant proteins using the Pichia pastoris system. However, the recent re-examination of our VrD1 expression system showed that some of the previous constructions contained unexpected base frame-shift. On the other hand, regarding the parts with correct sequences, the yields of the consequent protein purification were rather low. This may give rise to the difficulty in reaching sufficient sample size in the experiments of whole-cell recordings performed previously. Therefore, in the present study, we focus our work on two aspects. (1) Reconstruction of the appropriate VrD1 mutants with correct sequences. (2) Increasing the expression levels via optimized preparation of VrD1 mutants expressed in pichia. In the past, we have utilized G418-resistance as a selection marker for high gene copy numbers and potentially for a high expression level. However, this strategy could not ensure the satisfactory expression yields. In the present study, we combine the small-scale expression and the western blotting as checkpoints to improve the screening strategy by exclusion of the false-positive G418-resistant transformants. After transformation of the VrD1-pPIC9K plasmids for R26A and K24A mutants into P. pastoris (SMD1168), seven well-grown transformants on 0.5 mg/mL G418/YPD plates were selected for high copy number and high expression level strains. Using this screening strategy, we obtained Y38 and Y43 for the expression of VrD1-R26A. The western blot results demonstrated that the colony Y38 has significantly higher target protein expression than the colony Y43. Results from the large-scale expressions are in line with those from small-scale expressions. Moreover, using the site-directed mutagenesis, we have successfully generated VrD1-K7A from wild-type VrD1. This will be a convenient method in the preparation for other mutant strains with higher insecticidal and antifungal activities in the future. II. Investigation on the membrane target(s) of VrD1 in insect cells and the molecular mechanism of VrD1 insecticidal activity: Base on sequence and structural alignment, we have postulated that VrD1 may utilized a similar interaction mode as short scorpion toxins to act on insect cell membranes with K+-channel or Cl--channels as molecular targets. Preliminary data has excluded Cl--channels for candidates based on electrophysiological experiments. To study the protein-protein interaction for VrD1 and cell membranes, two major aspects need to be considered. 【1】 Generation of large amounts of VrD1 protein, at least a few milligrams for each experiment. Instead of using pichia expression system, we purify native VrD1 from mung bean seeds. About 5~6 milligrams of VrD1 protein can be produced from 500 grams of mung bean seeds. Mass spectrum has confirmed the correctness of VrD1. 【2】 Choice of appropriate cell-lines that allow VrD1 to interact with its membrane target(s). Previous reports showed that VrD1 can lead to a significant inhibitory effect on Sf21 or Sf9 (Spodoptera frugiperda 21 or 9). However, due to the lack of Spodoptera frugiperda genome database, it will be very difficult to identify unknown VrD1-target candidates from membrane extracts. Therefore, Drosophila S2 cell-line was chosen in this study to perform cell survival assay before the investigation with membrane proteomics. | en |
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dc.description.tableofcontents | 口試委員會審定書……..........................………………i
誌謝……………………………………........…………........ii 中文摘要.................................................iii 英文摘要…………………………...............................v 目錄…………………………….................................I 實驗圖表目錄..……………….……...........................IV 參考文獻附圖目錄..........................................V 縮寫檢索表……………………………..........................VI 第一章、前言…………………………………………………………….1 1. 植物防禦素 (Plant defensins)………………………………………………........…..1 (1) 植物防禦素之發現......................................................1 (2) 植物防禦素的結構特性與活性.......................................................1 2. 綠豆防禦素 (Vigna radiata defensin 1)...………......3 (1) 綠豆防禦素簡介.......................................3 (2) VrD1 的結構與序列性質..................................3 3. VrD1的研究進展與沿革....................................4 (1) VrD1在大腸桿菌系統中的表現..........................4 (2) 從綠豆VC 6089A 成熟種子中純化天然VrD1胜肽............5 (3) VrD1在嗜甲醇酵母菌 (Pichia pastoris) 中的表現........5 (4) VrD1序列性質比對.......................................6 4 綠豆防禦素的蛋白質活性分析...............................6 (1) 體外蛋白質合成的抑制活性...............................6 (2) 抑制真菌與細菌生長的活性...............................6 (3) 抗綠豆豆象及秋行軍蟲的活性.........................................................7 (4) 抑制昆蟲澱粉水解酶 (insect α-amylase) 的活性.........7 5. 綠豆防禦素具有兩群特殊的保守性鹼性殘基.......10 (1) 綠豆防禦素的兩群保守性鹼性殘基可能分別對VrD1的抗蟲與抗菌活性有重要影響性.....................................10 (2) 其中一群鹼性胺基酸可能與VrD1抗真菌、細菌的活性有關....10 (3) 另一群鹼性胺基酸群可能與抗綠豆豆象活性有關....11 (4) 蠍毒蛋白 (Scorpion toxins) 以及其他離子通道抑制性蛋白 (channel inhibitors ).....................................13 第二章、研究動機..........................................15 第三章、材料與方法........................................18 實驗材料..................................................18 質體......................................................18 菌株......................................................18 細胞株....................................................18 藥品......................................................18 儀器......................................................21 實驗方法..................................................22 1.構築綠豆防禦素VrD1表現質體........................................................22 (1) 取得表現質體pPIC9K-K24A VrD1及pPIC9K-R26A VrD1........22 (2) 構築表現質體 pPICZαA-K12A VrD1.......................22 2. 聚合酶連鎖反應 (Polymerase Chain Reaction;PCR)........23 3. 定點突變 (Site-directed mutagenesis) VrD1表現質體......23 4. 大腸桿菌之轉形 (Tranformation).........................24 5. E. coli (DH5α) 菌落聚合酶連鎖反應 (colony PCR).........25 6. 小量酵母菌 Pichia pastoris 培養法........................................................26 7. 酵母菌菌落聚合酶連鎖反應 (colony PCR)......................................................26 8. 電穿孔 (Electroporation) 轉型酵母菌........................................................27 (1) 製備酵母菌株勝任細胞 (Spheroplasting of P. pastoris)..27 (2) 酵母菌轉型反應........................................28 9. 醋酸鋰法轉型酵母菌........................................................29 (1) 製備酵母菌株勝任細胞 (Spheroplasting of P. pastoris)..29 (2) 酵母菌轉型反應........................................29 10. 酵母菌表現菌株之篩選........................................................30 (1) 挑選轉型成功之高抗藥性菌株 (pPICZ9K系統)..................................30 (2) 挑選轉型成功之高抗藥性菌株 (pPICZαA系統)..................................31 (3) 小量表現 (Small-scale expression) 篩選高表現量之酵母菌菌株........31 11. 大量表現---搖瓶培養........................................................32 12. 以FPLC系統與陽離子交換樹脂純化VrD1....................32 13. 自綠豆品系VC 6089A 綠豆種子萃取天然的綠豆防禦素.......34 14. 昆蟲細胞之培養........................................................37 (1) 秋行軍蟲細胞株 (Sf21 cell lines) 之培養........................................................37 (2) 果蠅細胞株 (Drosophila S2 cell lines) 之培養..........38 (3) 細胞冷凍..............................................38 (4) 細胞解凍..............................................38 15. 細胞存活率實驗 (Survival assay)....................................................39 16. SRB assay (Sulforhodamine B cytotoxicity assay) 計算細胞相對存活率................................................40 17. SDS-PAGE 膠體電泳法以及西方墨點法 (western blot)......41 18. 圓點墨點分析法 (Dot blot).............................44 19. 硝酸銀染色法 (Silver stain)....................................................44 20. 蛋白濃度測定法........................................................45 (1) Bradford method 蛋白濃度測定法........................................................45 (2) Lowry method 蛋白濃度測定法...........................45 第四章、結果與討論........................................47 I. 利用嗜甲醇酵母菌表現系統進行最適化綠豆防禦素VrD1突變株之製備.....................................................47 1. VrD1 突變株之VrD1-pPIC9K質體的建構與定序檢測..........47 2. R26A、K24A VrD1的表現株選殖、表現、與純化..............47 (1) R26A、K24A VrD1表現株的轉型與G418抗性篩選.............47 (2) 以小量表現篩選法尋找表現量最佳的表現株................47 (3) 誘導前菌液濃度對表現量的影響性論述...................48 (4) R26A VrD1表現株的大量表現及純化分析...................48 (5) 方法學上的進展與討論..................................49 3. K12A VrD1-pPICZαA的建構與表現.........................50 (1) 嘗試 pPICZαA 表現系統表現VrD1蛋白的原因..............50 (2) 將DNA序列優化後的K12A VrD1構築至pPICZαA質體..........51 (3) K12A VrD1*表現株之篩選與小量表現篩選法分析結果.......51 4. 以定點突變法重新建構剩餘的點突變VrD1質體...............51 II. 尋找綠豆防禦素VrD1在昆蟲細胞膜上之標的蛋白及其抗蟲機制探討.......................................................52 1. 以蛋白質體學方法尋找VrD1在昆蟲系統之標的蛋白策略簡述..52 2. Wild type VrD1的表現與純化...........................52 3. 由綠豆萃取與純化天然的綠豆防禦素 (native VrD1).......53 4. 昆蟲細胞株的選擇.....................................53 5. VrD1在50 μg/mL濃度下會造成果蠅細胞株的死亡......................................................54 6. 綠豆萃取之天然綠豆防禦素與大腸桿菌表現的VrCRPTSP之活性差異探討....................................................54 第五章、總結與未來展望...................................57 第六章、實驗圖表......................................58 附錄I、培養液配方........................................82 附錄II、參考文獻附圖..................................86 參考文獻........................................................91 實驗圖表目錄 圖1、 VrD1-pPIC9K質體建構...............................58 圖2、 本論文中預計製備的七株VrD1突變株總整:過去研究及本論文之進展..................................................59 圖3、 K6A, K7A, K12A VrD1經由定序檢測發現表現質體有frame-shift問題.................................................60 圖4、將線性化後的質體 K24A VrD1-pPIC9K 及 R26A VrD1-pPIC9K 送入P. pastoris SMD1168 中,獲得 His+ 轉型株............62 圖5、分別自K24A, R26A 轉型後的MD培養基上各挑出60株 His+ 轉型株,以5’ AOX - 3’ AOX primers進行Colony PCR....63 圖6、 K24A VrD1、R26A VrD1轉型株在 0.5 mg/mL G418/YPD 培養基上的生長情形...........................................64 圖7、 以 5 mL 小量培養的方式,配合western blot 分析七株候選表現株的果.............................................65 圖8、 VrD1表現株Y38及Y43大量表現後,純化及SDS-PAGE、western blot的分析結果顯示兩株表現株皆能成功表現R26A VrD1且,Y38比Y43能獲取更大量的產量.........................66 圖 9、Y38A及Y43A純化產物之濃度測定.................68 圖10、K12A* VrD1-pPICZαA之質體建構.....................69 圖11、K12A* VrD1-pPICZαA經由定序結果確認無誤..........70 圖12、小量表現分析western blot結果顯示K12A VrD1目前還沒有挑到可以表現目標蛋白的表現株................................71 圖13、以定點突變法重新建構剩餘的點突變VrD1質體...........72 圖14、以蛋白交互作用來尋找VrD1標的蛋白之策略示意圖.......73 圖15、Wild type VrD1之表現與純化結果.....................................................74 圖16、 由綠豆品系VC 6089A種子中萃取天然成熟胜肽VrD1純化結果 (A)....................................................75 圖17、 由綠豆品系VC 6089A種子中萃取天然成熟胜肽VrD1純化結果 (B)......................................................76 圖18、 由綠豆品系VC 6089A種子中萃取天然成熟胜肽VrD1純化結果-總結......................................................77 圖19、由綠豆萃取之天然綠豆防禦素VrD1,經質譜儀鑑定後,確定為VrD1無誤..........................................78 圖20、細胞存活實驗顯示VrD1在50 μg/mL濃度下會造成果蠅細胞株 (Drosophia S2 cells) 細胞膜破裂的現象....................79 圖21、細胞存活實驗顯示 VrD1在50 μg/mL 濃度下無法對 Sf21 細胞株造成明顯的影響......................................80 圖22、50 μg/mL VrD1處理後之昆蟲細胞細胞存活率統計結果....81 參考文獻圖表目錄 附錄圖1、具有綠豆豆象抗性的綠豆品系 VC 6089A 綠豆種子....86 附錄圖2、綠豆豆象 Callosobruchus chinensis 的演變及生活週期........................................................87 附錄圖3、VrCRPTSP 造成立枯絲核菌生長受抑制的現象.......88 附錄圖4、VrCRPTSP 加入秋行軍蟲細胞株Sf21 (Spodoptera frugiperda 21) 培養液中,濃度在1.7 μM 以上即會造成顯著細胞死亡......................................................89 附錄圖5、誘導E.coli M15表現全長VrCRP與VrCRPTSP 表現後所造成的宿者細胞溶菌或生長受抑制的現象.........................90 | |
dc.language.iso | zh-TW | |
dc.title | I. 利用嗜甲醇酵母菌表現系統進行最適化綠豆防禦素VrD1突變株之製備
II. 尋找綠豆防禦素VrD1在昆蟲細胞膜上之標的蛋白及其抗蟲機制之探討 | zh_TW |
dc.title | I. Optimized Preparation of VrD1 Mutants Expressed in Pichia pastoris
II. Investigation on the Membrane Target(s) of VrD1 in Insect Cells and the Molecular Mechanism of VrD1 Insecticidal Activity | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林長平(Chan-Pin Lin),陳慶三(Ching-San Chen),陳威戎(Wei-Jung Chen),鄭宇哲(Yu-Che Cheng) | |
dc.subject.keyword | 綠豆防禦素,植物防禦素,嗜甲醇酵母菌,全細胞電位鉗制量測法,抗蟲, | zh_TW |
dc.subject.keyword | VrD1,plant defensin,whole-cell voltage-clamp,Insecticidal, | en |
dc.relation.page | 95 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-19 | |
dc.contributor.author-college | 牙醫專業學院 | zh_TW |
dc.contributor.author-dept | 口腔生物科學研究所 | zh_TW |
顯示於系所單位: | 口腔生物科學研究所 |
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