Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/28297
標題: 探討Deinococcus ficus 基因群組 imuB-dnaE2 參與細胞突變之DNA修復機制及羽毛降解菌株之改良
Mechanism on the gene cassette imuB-dnaE2 mediated cell mutation and DNA repair in Deinococcus ficus and creation of superior feather degrading mutant
作者: 曾宥綋
Zeng, You-Hong
關鍵字: Deinococcus ficus
菩提奇異球菌
gene cassette imuB-dnaE2
feather degradation
Deinococcus grandis
基因群組imuB-danE2
羽毛分解
巨大奇異球菌
出版社: 土壤環境科學系所
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摘要: Abstract The function of gene cassette lexA-imuB-dnaE2 was identified as TLS polymerase (participates in DNA repair and cell mutation) in many bacteria. The gene cassette lexA-imuB-dnaE2 was also found in D. ficus wild type strain, CC-FR2-10T and indicated the homologous proteins of TLS polymerase may exist in other species of Deinococcus genus. The conserved imuB gene primers (cimuB primers) of Deinococcus genus were designed by aligning the ImuY protein sequence of D. deserti and ImuB protein sequence of D. ficus. The imuB gene of D. grandis has been amplified by the cimuB primers with PCR. After analyzing the ImuB sequence of D. ficus, D. grandis and ImuY sequence of D. deserti, the conserved protein sequence within Deinococcus genus has been found and showed the ImuB protein was deduced from Deinococcus ancestor. The function of ImuB and DnaE2 in D. ficus has been identified as TLS polymerase under UV stress by insertional inactivation method. The superior feather degrading mutant D. ficus ZG207 (relative keratinolytic activity is two fold compared with stain CC-FR2-10T) and the inferior mutant D. ficus ZG227 (relative keratinolytic activity is half compared with strain CC-FR2-10T) were isolated after UV exposure. The zymogram profile of extracellular proteins in D. ficus ZG227 showed deficient protease activity of four extracellular proteins caused by UV-induced gene mutation. D. ficus ZG207 has high efficiency of feather degradation in initial growth stage, evidence of 64.8% of feather degradation and releasing a lot of various amino acids by incubating D. ficus ZG207 at 37℃ in NBHF medium (pH 9 and 0.75% feather) to the second day. The KerA protein of D. ficus was identified as keratinase by transfer the kerA gene of D. ficus to D. grandis DSM 3963 (transferred strain is D. grandis K76-1), the kerA gene was expressed in D. grandis K76-1, and then the relative keratinolytic activity and feather degradation percentage was increased to 2-3 fold compared with non-transferred wild type strain DSM 3963.
摘要 前人研究指出細菌之lexA-imuB-dnaE2 基因群組的功能為具有TLS (translesion synthesis) polymerase 的活性,即參與DNA 修復並造成細胞突變的發生。本研究發現Deinococcus ficus CC-FR2-10T之基因組中具有基因群組lexA-imuB-dnaE2,顯示Deinococcus 菌屬中之不同菌種可能具有TLS polymerase 的同源蛋白質。本研究經比對D. deserti 之ImuY 及D. ficus 之ImuB 蛋白質序列後,設計出Deinococcus 菌屬之高保留imuB基因引子 (即cimuB 引子), 並以PCR 放大出D. grandis 之imuB 基因,經由分析D. ficus 及 D. grandis 之ImuB與D. deserti 之ImuY蛋白質序列後,發現Deinococcus 菌屬之ImuB 蛋白質具有高的菌屬序列保留特性,顯示Deinococcus 菌屬之不同菌種的ImuB 蛋白質是經由Deinococcus祖先演化而來。本研究以插入破壞基因法 (insertional inactivation) 論證出D. ficus 的ImuB 及DnaE2 蛋白質在紫外光逆境下具有TLS polymerase 的活性。本研究另以紫外光照射D. ficus 野生菌株 CC-FR2-10T後,產生羽毛分解效能變好之突變菌株D. ficus ZG207,其相對角蛋白酶活性為野生菌株的2倍,及效能變差之突變株D. ficus ZG227,其相對角蛋白酶活性為野生菌株的一半。蛋白質活性染色 (zymogram) 圖譜顯示D. ficus ZG227 失去4個具有蛋白質酶活性的胞外蛋白質,顯示紫外光照射可導致D. ficus 之基因變異。D. ficus ZG207 培養於37℃之NBHF 羽毛培養基 (pH 9,0.75% 羽毛) 至第2天的羽毛分解率為64.8 % 並釋出多種胺基酸,顯示其具有在生長初期快速分解羽毛的能力。本研究經由轉移D. ficus 的kerA 基因至D. grandis DSM 3963 (轉殖菌株D. grandis K76-1) 後,D. grandis K76-1 表現D. ficus 之kerA 基因並提高其相對角蛋白酶活性及羽毛分解率約為非轉移菌株DSM 3963之2-3倍,進而論證出D. ficus 之 KerA 蛋白質具有角蛋白酶之活性。
URI: http://hdl.handle.net/11455/28297
其他識別: U0005-0904201211030100
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0904201211030100
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