百合防禦相關蛋白LsGRP1之胜肽片段抗菌活性與機制探討

Abstract

抗菌胜肽具有抗菌活性，由12~50 個胺基酸組成，廣泛存在於各種生物體如人類、昆蟲、植物、細菌等，可以協助生物體抵抗細菌、真菌、病毒、寄生蟲等各種病原感染。LsGRP1 (Lilium ‘Star Gazer’ glycine-rich protein)是一源自於葵百合的防禦相關蛋白，由138 個胺基酸組成；C 端序列的16%為半胱胺酸(cysteine)，具有良好的抑菌能力，為一抗菌胜肽。為了解LsGRP1 的應用潛力，測試其對臺灣多種常見植物病原菌的抑制作用，發現LsGRP1 的C 端胜肽(LsGRP1C)可有效抑制Alternaria brassicicola、Botrytis cinerea 與Colletotrichum acutatum 等真菌孢子發芽，對Pseudomonas syringae pv. syringae、Xanthomonas campestris pv.vesicatoria 與Clavibacter michiganensis 等細菌則有致死的作用。於植體上，LsGRP1C 可在甘藍葉片及草莓花瓣分別抑制黑斑病及灰黴病的發展，並可抑制P. syringae pv. syringae 在番茄葉片上的感染。進一步探討LsGRP1C 的抑菌機制，以掃描式電子顯微鏡觀察細菌細胞外部形態的變化，發現處理LsGRP1C 的細菌表面有疣狀凸起並於細菌周圍出現許多球狀物；藉由螢光染劑SYTOX green 觀察LsGRP1C 對細菌細胞的影響，發現此抗菌胜肽可造成細菌細胞膜穿孔。

Antimicrobial peptides (AMPs) are a class of small host defense peptides, normally 12 to 50 amino acids, with antimicrobial activity against bacteria, fungi, viruses, parasites and other pathogenic microorganisms on humans, insects, plants, etc. LsGRP1 (Lilium ‘Star Gazer’ glycine-rich protein) is a plant defense-related protein derived from Lilium, consisting of 138 amino acids with 16% cysteine in C-terminal region. Antimicrobial activity of the synthetic C-terminal LsGRP1 (LsGRP1C) had been shown previously. To understand the potential applications of this peptide, its inhibitory abilities to a variety of plant pathogens were assayed and found to inhibit spore germination of Alternaria brassicicola, Botrytis cinerea, and Colletotrichum acutatum. In addition, this peptide caused bacterial cell death in Pseudomonas syringae pv. syringae, Xanthomonas campestris pv. vesicatoria, and Clavibacter michiganensis., In planta assays showed that this synthetic LsGRP1C inhibited symptom developments of cabbage black spot, strawberry gray mold, and tomato bacterial leaf spot. For investigation of the antibacterial mechanism, bacterial cell surface was examined by scanning electron microscopy, and the results showed that LsGRP1C caused protrusion of bacterial surface and formation of spherical bodies aside of bacterial cells. Examination with fluorescent dye SYTOX green showed that this AMP could increase membrane permeability of the target bacterial cells.