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

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。

關鍵字

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

並列摘要

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.

並列關鍵字

VrD1 ； plant defensin ； whole-cell voltage-clamp ； Insecticidal

參考文獻

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

Broekaert WF, Terras FR, Cammue BP, Osborn RW (1995) Plant defensins: novel antimicrobial peptides as components of the host defense system. Plant Physiol

Cabral KM, Almeida MS, Valente AP, Almeida FC, Kurtenbach E (2003) Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inefficiency of the STE13 protease. Protein Expr Purif 31(1):

Catterall WA (1980) Neurotoxins that act on voltage-sensitive sodium channels in

excitable membranes. Annu Rev Pharmacol Toxicol 20: 15-43

國際替代計量

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

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