已發現的抗菌胜肽大多具分子量小於10 kDa、偏疏水性、具細胞膜活性且總帶電性多為正電等性質，文獻鮮少報告源自海洋矽藻的抗菌胜肽，本研究從底棲性矽藻菱形藻細胞外蛋白質分離出一新的胜肽並命名為ABMOK1，為由12個胺基酸組成的胜肽鏈，序列為CGYCGACVGVCK，由SDS-PAGE小於6.5 kDa分子量的蛋白質中取得，以液相層析串聯質譜儀(LC-MS-MS)分析其蛋白質數量及序列，總共獲得336段胜肽，其中6段是具有潛在抗菌胜肽活性，利用縮小一般抗菌胜肽特徵挑選出來的候選胜肽，其特徵包括10-15個胺基酸的胜肽鏈、疏水性胺基酸百分率大於等於0.53且小於等於0.6及總帶電性大於等於+1且小於等於+3。ABMOK1是從這6段胜肽鏈中所任意挑出的一段。掃描式電子顯微鏡(SEM)觀察結果顯示，濃度100 g/mL時ABMOK1，Escherichia coli細胞膜嚴重受到破壞，與大腸桿菌細胞膜進行交互作用，具有大腸桿菌細胞膜活性的特性，對照抗菌胜肽作用模型，ABMOK1抗菌機制屬於清潔劑模式，在同樣條件在下，Staphylococcus aureus就沒有此現象，這也說明了ABMOK1具有選擇性的進行分子交互作用。

Most of antimicrobial peptides (AMPs) are less than 10 kDa in molecular weight, hydrophobic, membrane active, and have an overall net positive charge. To date, few AMPs have been isolated from marine diatoms. This research studied novel AMPs from marine benthic diatoms and examined their antimicrobial activity. Extracellular extracts of the benthic diatom Nitzschia sp. 1 CCL-2013 were analyzed; the major fraction was less than 6.5 kDa of molecular weight. LC-MS/MS analysis on this major fraction identified 336 peptides. Out of the 336 peptides, 6 potential AMPs were identified using the following strigent critera: being 10 to 15 aa in length, having more than positive one to three in total net charge and at percentage of hydrophobic residues between 0.53 and 0.6. One peptide which was named as ABMOK1 was arbitrarily picked from the 6 peptides and tested for its anbitiotic activity. This novel AMP, ABMOK1, is characterized by the sequence of CGYCGACVGVCK. The SEM of images of Escherichia coli treated by 100 g/mL of ABMOK1 showed that holes were punched on the bacterial membrane, suggesting that ABMOK1 is a membrane-active antibacterial peptide which takes a detergent-like mode of destructive action.

審定書 i
致謝詞 ii
中文摘要 iii
Abstract iv
目錄 v
一、緒言 1
二、菱形藻採樣、分離及鑑定 5
2.1 摘要 5
2.2 前言 6
2.3 研究方法 8
2.3.1 採樣及矽藻培養 8
2.3.2 掃描式電子顯微鏡影像擷取及前處理 8
2.3.3 矽藻科內屬的檢索表 9
2.3.4 去氧核糖核酸萃取及片段放大 9
2.3.5 建構親緣關係演化樹及樣本與10種相近種之成對距離 9
2.4 結果與討論 10
三、 菱形藻細胞外短鏈胜肽純化與定序 20
3.1 摘要 20
3.2 前言 21
3.3 研究方法 24
3.3.1 掃描式電子顯微鏡影像擷取及前處理 24
3.3.2 蛋白質萃取 24
3.3.3 胜肽分離 24
3.3.4 質譜分析胜肽序列 26
3.4 結果與討論 26
四、 抗微生物胜肽之統計分析 35
4.1 摘要 35
4.2 前言 36
4.3 研究方法 37
4.3.1 估計母體比例之樣本數選擇 37
4.3.2 樣本來源 38
4.3.3 統計分析 38
4.4 結果與討論 38
五、 ABMOK1的抗菌性檢測 50
5.1 摘要 50
5.2 前言 51
5.3 研究方法 55
5.3.1 Nitzschia sp. 1 CCL-2013胜肽之篩選 55
5.3.2 ABMOK1胜肽對細菌細胞影響檢測 55
5.3.3 掃描式電子顯微鏡影像擷取及前處理 56
5.4 結果與討論 56
六、 總結論 69
七、 參考文獻 70
八、附錄 75
一、本研究所分離的短鏈胜肽 75
二、本論文研究期間發表之論文 89
九、個人履歷 99

林惠玲，陳正倉 (2000)：統計學－方法與應用。台北市：雙葉書廊。
Arouri A., Dathe M., and Blume A. (2009) Peptide induced demixing in PG/PE lipid mixtures: A mechanism for the specificity of antimicrobial peptides towards bacterial membranes? Biochimica et Biophysica Acta. 1788:650-659.
Bates S.S., Gaudet J., Kaczmarska I., and Ehrman J.M. (2004). Interaction between bacteria and the domoic-acid-producing diatom Pseudo-nitzschia multiseries (Hasle) Hasle; can bacteria produce domoic acid autonomously? Harmful Algae. 3:11-20.
Brandenburg L.O., Merres J., Albrecht L.J., Varoga D., and Pufe T. (2012). Antimicrobial peptides: multifunctional drugs for different applications. Polymers. 4:539-560.
Carpizo-Ituarte E., and Hadfield M.G. (1998). Stimulation of metamorphosis in the polychaete Hydroides elegans Haswell (Serpulidae). The Biological Bulletin. 194:14-24.
Creighton T.E. (1992) Proteins: structures and molecular properties. Freeman, New York.
Dobretsov S., Dahms H.U., and Qian P.Y. (2006). Inhibition of biofouling by marine microorganisms and their metabolites. Biofouling. 22:43-54.
Gell P., and Sonneman J. (2012). Monash University: key to the common general freshwater diatoms. Retrieved October 02, 2013, from http://arts.monash.edu.au/ges/research/cpp/diatoms/generic.php.
Hachem F., Andrews, B. and Asenjo J. (1996). Hydrophobic partitioning of proteins in aqueous two-phase systems. Enzyme and Microbial Technology. 19:507-517.
Hasle G.R., Syvertsen E.E., Steidinger K.A., Tangen K., and Tomas C.R. (1996). Identifying marine diatoms and dinoflagellates. Academic Press, New York.
Huang S., and Hadfield M.G. (2003). Composition and density of bacterial biofilms determine larval settlement of the polychaete Hydroides elegans. Marine Ecology. 260:161–172.
Kamp R.M., Calvete J.J., and Choli-Papadopoulou T. (2004). Methods in proteome and protein analysis. Springer, New York.
Krämer R., and Jung K. (2010). Bacterial Signaling. Wiley, Hoboken.
Lu M., and Tjerneld F. (1997). Interaction between tryptophan residues and hydrophobically modified dextran: Effect on partitioning of peptides and proteins in aqueous two-phase systems. Journal of Chromatography A. 766:99-108.
Lundholm N., and Jvind Moestrup Ø. (2000). Morphology of the marine diatom Nitzschia navis-varingica, sp. nov. (Bacillariophyceae), another producer of the neurotoxin domoic acid. Journal of Phycology. 36:1162-1174.
Molino P.J., Childs S., Eason Hubbard M.R., Carey J.M., Burgman M.A., and Wetherbee R. (2009a). Development of the primary bacterial microfouling layer on antifouling and fouling release coatings in temperate and tropical environments in Eastern Australia. Biofouling. 25:149-162.
Molino P.J., Campbell E., and Wetherbee R. (2009b). Development of the initial diatom microfouling layer on antifouling and fouling-release surfaces in temperate and tropical Australia. Biofouling. 25:685-694.
Mori T., O'Keefe B.R., Sowder R.C., Bringans S., Gardella R., Berg S., Cochran P., Turpin J.A., Buckheit R.W., and McMahon J.B. (2005). Isolation and characterization of griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp. Journal of Biological Chemistry. 280:9345-9353.
Pool J.R., Kruse N.A., and Vis M.L. (2013). Assessment of mine drainage remediated streams using diatom assemblages and biofilm enzyme activities. Hydrobiologia. 709:101-116.
Prigge S.T., Kolhekar A.S., Eipper B.A., Mains R.E., and Amzel L.M. (1997). Amidation of bioactive peptides: the structure of peptidylglycine α-hydroxylating monooxygenase. Science. 278:1300-1305.
Rabilloud T. (2000). Proteome research: two-dimensional gel electrophoresis and identification methods. Springer, New York.
Reddy K., Yedery R., and Aranha C. (2004) Antimicrobial peptides: premises and promises. International Journal of Antimicrobial Agents. 24:536-547
Rendueles O., Beloin C., Latour-Lambert P., and Ghigo J. (2014). A new biofilm-associated colicin with increased efficiency against biofilm bacteria. The International Society for Microbial Ecology Journal. 8:1275-1288.
Salta M., Wharton J.A., Blache Y., Stokes K.R., and Briand J.F. (2013). Marine biofilms on artificial surfaces: structure and dynamics. Environmental Microbiology. 15:2879-2893.
Sewald N., and Jakubke H.D. (2009a). Peptides: Chemistry and Biology. In Biology of Peptides, 2nd Edition. Wiley, Hoboken.
Sewald N., and Jakubke H.D. (2009b). Peptides: Chemistry and Biology. In Fundamental Chemical and Structural Principles, 2nd Edition. Wiley, Hoboken.
Siuzdak G. (1996). Mass spectrometry for biotechnology, 1st Edition. Academic Press, New York.
Spaulding S., and Edlund M. (2008) Nitzschia sp. In Diatoms of the United States. Retrieved October 02, 2013, from http://westerndiatoms.colorado.edu/taxa/genus/Nitzschia.
Steiner H., Hultmark D., Engström Å., Bennich H., and Boman H. (1981). Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature. 292: 246-8.
Tamura K., Stecher G., Peterson D., Filipski A., and Kumar S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular biology and evolution. 30:2725-2729.
Waghu F.H., Gopi L., Bara, R.S., Ramteke P., Nizami B., and Idicula Thomas S. (2014). CAMP: Collection of sequences and structures of antimicrobial peptides. Nucleic Acids Research. 42:1154-1158.
Wang G., Li X., and Wang Z. (2009). APD2: the updated antimicrobial peptide database and its application in peptide design. Nucleic Acids Research. 37:933-937.
Wang R., and Chait B.T. (2009). Protein ladder sequencing. In The Protein Protocols Handbook, 3rd Edition. Springer, New York.
White T.J., Bruns T., Lee S., and Taylor J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: a Guide to Methods and Applications. Academic Press, New York.
Wimley W.C. (2010). Describing the mechanism of antimicrobial peptide action with the interfacial activity model. American Chemical Society Chemical Biology. 5:905-917.
Wustman B.A., Gretz M.R., and Hoagland K.D. (1997). Extracellular matrix assembly in diatoms (Bacillariophyceae)(I. A model of adhesives based on chemical characterization and localization of polysaccharides from the marine diatom Achnanthes longipes and other diatoms). Plant Physiology. 113:1059-1069.
Wustman B.A., Lind J., Wetherbee R., and Gretz M.R. (1998). Extracellular matrix assembly in diatoms (Bacillariophyceae). Plant Physiology. 116:1431-1441.
Zardus J.D., Nedved B.T., Huang Y., Tran C., and Hadfield M.G. (2008). Microbial biofilms facilitate adhesion in biofouling invertebrates. The Biological Bulletin. 214:91–98.