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標題: Functional analysis of the low-molecular-weight bacteriocin, Carocin S4, from Pectobacterium carotovorum subsp. carotovorum
Pectobacterium carotovorum subsp. carotovorum 低分子量細菌素 Carocin S4 之功能探討
作者: 胡家銘
Hu, Jia-Ming
關鍵字: soft-rot disease
出版社: 化學系所
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摘要: Pectobacterium carotovorum subsp. carotovorum (Pcc) causing the soft-rot disease of many commercially important plants is a Gram-negative phytopathogenic enterobacterium. It causes very great economic loss, but there is no effective method to control the disease. Bacteriocins produced from bacteria are proteinaceous compounds and have bacteriostatic or bactericidal activities against bacteria species that are closely related to the producing strain. With the advantage of low toxicity to environment, bacteriocins are chosen as natural bactericides to prevent phytopathogenic infection of plants. We hope to find an effectual method to control soft rot by studying bacteriocins produced from Pcc (rif-TO6) . Carocin S4, a chromosome-encoded low-molecular-weight bacteriocin (LMWB) with DNase activity, is produced from a Pcc strain, rif-TO6. The carocin S4 gene includes 2750-bp and contains two open reading frames (ORFs). The former ORF (ORF1) containing 2484-bp is responsible for a toxic protein (killer protein, 89.9kD), we named it as caroS4K. The later ORF (ORF2) containing 270-bp is responsible for a immunity protein (10.2kD), we named it as caroS4I. Interestingly, ORF2 overlaps by 4-bp with ORF1. In this study, overproduction, purification and characterization in vitro of Carocin S4 protein were reported. To determine the contents of metal elements in the CaroS4K protein, CaroS4K was assayed by ICP-Mass. The result showed that the major elements in the CaroS4K were Ca2+, Mg2+, Cu2+, and Zn2+ ions. The metal-dependent DNase activity of CaroS4K was assayed by using pMCL200 plasmid DNA as a substrate with Ca2+, Mg2+, Cu2+ and Zn2+ ions. The result showed that Ca2+, Mg2+ and Zn2+ ions were able to enhance the activity of CaroS4K. Besides, the DNase activity of CarocS4K was assayed by using pMCL200 plasmid DNA as a substrate by various temperatures. The result showed the optimal temperature for the DNase activity of CaroS4K was about 50℃.
Pectobacterium carotovorum subsp. carotovorum (Pcc) 是一種會造成許多經濟作物得到細菌性軟腐病的革蘭氏陰性病原菌。目前仍找不到有效的方法防治此疾病,所以 Pcc 造成經濟上嚴重的損失。細菌素是一種由細菌分泌的蛋白類化合物,能夠抑制或殺死與生產菌種有緊密關係的親源菌種。由於細菌素有對環境低毒性的優點,所以常被選為預防植物細菌性感染的天然殺菌劑。希望透過研究由 Pcc 菌種 rif-TO6 所生產的低分子量細菌素,找到有效控制細菌性軟腐病的方法。 Carocin S4 是一種由 Pcc 菌種 rif-TO6 所生產的細菌素蛋白,此蛋白是由染色體所編碼且含有水解 DNA 活性。 Carocin S4 基因全長為2750-bp 且包含了兩個開放閱讀框架 (ORFs) :前面的開放閱讀框架 (ORF1) 全長 2484-bp,生產毒性蛋白,命名為 caroS4K ; 後面的開放閱讀框架 (ORF2) 全長 270-bp,生產免疫蛋白,命名為 caroS4I。有趣的是 ORF2 與 ORF1 重疊4-bp。 在本次研究中,探討有關 Carocin S4 蛋白的大量表現、純化以及 in vitro特性。將 CaroS4K 蛋白經由感應偶合電漿質譜儀 (ICP-MS) 檢測金屬含量,結果發現 CaroS4K 蛋白主要含有的金屬為 Ca2+, Mg2+, Cu2+ 和 Zn2+ 離子。利用質體 DNA (pMCL200) 當受質,分別探討 Ca2+, Mg2+, Cu2+ 和 Zn2+ 離子,對 CaroS4K 的活性有何影響。結果顯示Ca2+, Mg2+ 和 Zn2+ 離子,可以提升 CaroS4K 水解 DNA 的活性。另外,利用質體 DNA (pMCL200) 當受質,在不同的溫度下與 CaroS4K 蛋白反應。結果顯示CaroS4K 蛋白水解 DNA 的活性,最佳溫度約為50℃。
其他識別: U0005-2208201116241900
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