Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/28070
標題: Deinococcus ficus CC-FR2-10T抗UV輻射特性研究
Characterization of UV resistance in Deinococcus ficus CC-FR2-10T
作者: 曾宥綋
Zeng, You-Hong
關鍵字: Deinococcus ficus CC-FR2-10T
菩提奇異球菌
UV
antibiotic rifampicin
mutation
紫外光
抗生素rifampicin
突變
出版社: 土壤環境科學系所
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摘要: Deinococcus ficus CC-FR2-10T isolated from papal Ficus religiosa L. rhizosphere in Taiwan. This bacterium is a novel species. Deinococcus radiodurans R1 had resistant ability to ultraviolet, ionizing radiation and chemical compound which may cause damage to DNA, so the DNA repair system in this bacterium was well studied. There is no error prone SOS DNA repair system found in D. radiodurans after exposure to UV radiation. Strain R1 can survive under acute exposures to gamma radiation and ultraviolet without undergoing induced mutation. Many species in the genus Deinococcus were isolated from various environment but the comparison of DNA repair system in each species has not been done before. In the present study, mutation of D. ficus was monitored after exposure to UV and the mutation rate was calculated by changes of resistance to rifampicin. The results showed that mutation rate in D. ficus was 21.6% but there was only 0.42% in D. radiodurans after exposure to same lethal UV condition. It was proposed that UV radiation will induce SOS DNA repair system in D. ficus. After sequencing and alignment of rpoB gene among D. ficus wild type and mutant ZgR2, two amino acids substitution occurred. A amino acid changes from proline to leucine in the rif region was found, which escape the attacking by antibiotic rifampicin . D. ficus mutant ZgR2 was mutated again by UV radiation to assess the resistant ability of the mutant. The results revealed that UV exposure caused mutation of D. ficus. D. ficus mutant ZgTe1-7 can grow at 50℃ while there is no growth in wild type strain. After exposure to UV for 1 hour, the survival rate of D. ficus wild type strain was 1 % but there was only 0.1% in mutant ZgTe2, 5, 6, 7. When I aligned the RecA sequence between D. ficus and D. radiodurans, found D. ficus RecA sequence was core domain. Core domain in RecA sequence which binds to single strand DNA and influence RecA filament production was amplified by PCR and sequenced. The results showed that there was no mutation in the core domain of mutant ZgTe2, 5, 6, 7. D. ficus mutant ZgTe1 can grow at 55℃ incubator, mutant ZgTe3 can grow at 3 % NaCl NB and ZgTe5 can grow at pH 4 NB. The recA gene expression of D. ficus CC-FR2-10T was monitored under normal and UV exposure (15 min) condition by reverse transcription assay. Expression of recA gene increase after UV exposure might indicate SOS response in D. ficus CC-FR2-10T.
菩提奇異球菌 (Deinococcus ficus CC-FR2-10T) 是分離自菩提樹 Ficus religiosa L. 根圈之臺灣本土新菌種。 Deinococcus radiodurans具有抵禦紫外光、游離輻射及會造成 DNA 傷害藥劑之能力,因此其DNA修復機制被廣泛的研究。D. radiodurans DNA受傷時並無明顯表現SOS DNA修復系統,以進行DNA修復,此外亦不易因為紫外光或游離輻射之照射而發生突變作用。Deinococcus 菌屬中陸續有許多新菌種的發現,但新菌種DNA受傷害所表現之修復方式則很少人研究。本研究評估以紫外光照射 D. ficus 是否容易誘發突變作用,及對於經由照射後產生之突變株進行研究。本研究以菌株抗抗生素rifampicin之能力進行評估是否紫外光誘導菌株表現SOS SNA 修復系統,試驗結果發現D. ficus 照射紫外光達1% 存活率下,其突變株能抵禦抗生素之比例為21.6% 而D. radiodurans 則為0.42%,推論紫外光具有誘導D. ficus SOS DNA 修復系統的表現。 D. ficus ZgR2 rpoB 基因定序結果發現突變作用導致兩個胺基酸的改變,其中rif 區域中的proline轉變成leucine,許多文獻中皆指出此一改變能使得RNA聚合酶免於rifampicin的攻擊。此外以能抵禦rifampicin之突變株ZgR2進行再突變實驗,結果發現照射紫外光有刺激突變作用發生之現象。D. ficus RecA 蛋白質定序後之胺基酸序列與D. radiodurans RecA 序列相比為核心區域 (core domain),此核心區域為RecA 蛋白質與單股DNA鍵結的區域。本研究以紫外光照射獲得之突變株ZgTe1-7具有在50℃生長箱中生長的能力,但其照射紫外光1小時後突變株ZgTe2 、ZgTe5 、ZgTe6 、ZgTe7 之存活率接近0.1% 與原本的菌株 1% 相比明顯降低。突變株ZgTe2、5、6、7之RecA 核心區域 (core domain) 序列比對後並無發現任何的突變發生。此外突變株ZgTe1可於55℃生長箱中生長,ZgTe2則能於含有3% 氯化鈉鹽度的NB培養基中生長,而ZgTe5 則可於pH 4 之NB 培養基中生長。反轉錄 PCR 結果顯示 D. ficus recA 基因於細胞正常生長具有表現,若照射紫外光15分鐘後則表現量有增加的趨勢。本研究結果認為紫外光能造成D. ficus 突變作用的發生,且推論其DNA受傷後之修復方式與 D. radiodurans 之修復方式明顯不同。
URI: http://hdl.handle.net/11455/28070
其他識別: U0005-0708200712450500
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