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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭秋萍 | |
| dc.contributor.author | Yun-Lu Lai | en |
| dc.contributor.author | 賴芸璐 | zh_TW |
| dc.date.accessioned | 2021-05-20T21:11:57Z | - |
| dc.date.available | 2021-05-20T21:11:57Z | - |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-02-14 | |
| dc.identifier.citation | 陳巧燕。2009。未知功能 DUF28 protein 及色胺酸調控途徑在酵母菌氧化逆境反應之功能分析。國立台灣大學植物科學研究所碩士論文。
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10227 | - |
| dc.description.abstract | 除了在細菌之外， DUF28 蛋白質家族廣泛存在各物種，但對其功能所知甚少，僅知此蛋白在 Pseudomonas aeruginosa 中參與 swarming 泳動性與調節菌量感應反應 (quorum sensing)，在哺乳類動物中則扮演 COX I 的轉譯活化子 (translation activator) 的角色。本研究發現部分細菌具有兩個 DUF28 蛋白質，十分特別，故針對大腸桿菌之兩個 DUF28 蛋白 YeeN 及 YebC 進行功能分析，尤著重在氧化逆境反應之功能。序列發現在不同菌種的胺基酸序列相似度不盡相同，推測此蛋白質保有相似的立體結構。突變株特性分析結果顯示 ΔyeeN 及 ΔyebC 之泳動能力都有明顯增加，但唯有 ΔyeeN 抵抗 paraquat 氧化逆境的耐受度有明顯增加，互補實驗亦進一步證實這些結果；此外， YeeN 量的累積與逆境因子易造成此蛋白質在細胞質中呈點狀分布，而 YebC 除在生長死亡期會有段狀分佈的情況外，大多均勻分佈；啟動子分析結果顯示， yeeN 具有自己的啟動子且會受氧化逆境的抑制，而 yebC 啟動子則在細胞生長後期才具活性且不會受到氧化逆境的影響。進一步分析可能調節 yeeN 表現量之上游基因發現， recA 在氧化逆境下可調節 yeeN ； relA 在正常情況下可正向調控 yeeN ，但並未直接參與 yeeN 在氧化逆境條件之調控； hns 於生長後期會抑制 yeeN ，並可能參與 yeeN 負調控鞭毛合成相關基因之功能。綜合以上結果可知，大腸桿菌中 YeeN 與 YebC 皆可調節細菌之泳動力，但目前確認 YeeN 也同時參與氧化逆境反應，本研究首次報導兩個同為 DUF28 蛋白質家族成員之基因，彼此功能有同也有異。 | zh_TW |
| dc.description.abstract | DUF28 protein family ubiquitously exists in all organisms, except Archaea; however, information on their function is very limited. Previous studies demonstrated that DUF28 is involved in Pseudomonas aeruginosa swarming motility and quorum sensing, and functions as a translational activator of COX I in mammals. Interestingly, this study revealed the existence of two DUF28 homologs in some eubacteria. The aim of this study was to investigate functions of the two E. coli DUF28 proteins, namely YeeN and YebC, particularly their function in oxidative stress responses. Sequence comparisons revealed that bacterial DUF28 proteins shared low similarity, implying these proteins may preserve conserved tertiary structure. Null mutants ΔyeeN and ΔyebC both displayed increased swarming and swimming activities, while only ΔyeeN conferred increased tolerance to paraquat. Complementation assays further confirmed functions of these two proteins in motility and paraquat tolerance. Promoter analysis demonstrated that yeeN has its own promoter that can be repressed by oxidative stress. yebC promoter functioned only later growth phase and was not regulated by the test oxidative stress treatments. Protein localization analysis showed that accumulation and stress conditions could lead to polar distribution of YeeN in cytosol, while YebC uniformly distributed in cytosol and became polar distribution in a fragmented manner only at the death growth phase. Furthermore, by investigating the putative regulatory genes that may be involved in yeeN regulation, this study showed that recA could regulate yeeN under oxidative stress treatment. relA could upregulate yeeN expression under normal condition, but may not be involved in yeeN regulation under oxidative stress. On the other hand, hns could downregulate yeeN expression in late growth phase, and may regulate yeeN in flagella biosynthesis. Taken together, these results indicate that YeeN and YebC both function in bacterial motility, while YeeN also plays an important role in oxidative stress responses. This study is the first to uncover common as well as distinct functions of two bacterial DUF28 proteins. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-20T21:11:57Z (GMT). No. of bitstreams: 1 ntu-100-R97b42011-1.pdf: 3266188 bytes, checksum: 7b069b6ff33e28184933aebee0a8b7a0 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
謝誌 ii 中文摘要 iii Abstract iv 常用名詞之縮寫與全名對照表 vi 目次 vii 表目次 x 圖目次 xi 附錄目次 xii 第一章 前言 1 I. 大腸桿菌 1 II. 氧化逆境 1 III. 活性氧化物質 (reactive oxygen species, ROS) 的生合成及來源 2 IV. 大腸桿菌氧化逆境調控機制 3 1. OxyR Regulon 3 2. SoxRS Regulon 4 3. KatF (RpoS) Regulon 5 V. 鐵與鐵硫錯化合物對於氧化逆境調節之影響 5 VI. 大腸桿菌之泳動能力與鞭毛構造 6 VII. 大腸桿菌鞭毛的生合成及調控機制 8 VIII. DUF28 ( Domain of Unknown Function 28 ) 蛋白家族與相關研究 9 IX. 研究目標 10 第二章 材料與方法 11 I. 供試菌株、質體及菌株培養條件 11 II. 大腸桿菌野生株與突變株生物特性分析 11 1. 生長曲線分析 11 2. 生物膜形成分析 (biofilm formation assay) 11 3. 泳動能力測定 ( swimming and swarming motility ) 12 4. 逆境反應分析 12 III. YeeN 及 YebC 蛋白質分析 12 1. DUF28 蛋白質於大腸桿菌中表現位置之分析 12 2. western blotting 13 IV. 構築互補質體及 Promoter fusion lacZ 質體 14 1. 萃取大腸桿菌 genomic DNA 14 2. 聚合酶連鎖反應 (polymerase chain reaction, PCR) 15 3. 限制酶消化酵素水解及膠體純化 15 4. 載體與目標片段接合 15 5. 大腸桿菌熱休克轉型作用 16 6. 萃取大腸桿菌質體 DNA 16 7. 製備大腸桿菌電穿孔勝任細胞 16 8. 大腸桿菌電穿孔轉型作用 17 V. 大腸桿菌 RNA 樣本置備 17 1. 收取大腸桿菌 RNA 樣本 17 2. 大腸桿菌 RNA 萃取 17 3. 1% 甲醛電泳膠體置備 18 4. 反轉錄酶反應 18 5. 即時定量聚合酶連鎖反應 19 VI. yeeN 及 yebC 啟動子活性分析 19 VI. 生物統計分析 ( Statistic analysis ) 20 第三章 結果 21 I. 生物資訊功能性分析 21 1. DUF28 蛋白質在不同物種間之演化樹分析與蛋白質 domain 分析 21 2. 不同菌種間 DUF28 同源性蛋白質及演化樹之分析與胺基酸序列之比較 21 3. 大腸桿菌 yeeN 及 yebC 基因於微陣列資料庫中基因表現 22 II. 大腸桿菌突變株 ΔyeeN 及 ΔyebC 之生物特性分析 23 1. 生長曲線、生物膜形成及泳動能力測定 23 2. 逆境耐受性測試及相關基因表現 23 III. 驗證 yeeN 及 yebC 基因功能 24 1. 互補株之分析 24 IV. YeeN 及 YebC 蛋白質表現位置之觀察 25 1. YeeN 蛋白質表現位置及分布情形 25 2. YebC 蛋白質表現位置及分布情形 26 V. yeeN 、 yebC 之 operon 組成與調控 27 1. yeeN 之 operon 組成與調控 27 2. yebC 之 operon 組成與調控 28 VI. 上游可能參與調控 yeeN 或 yebC 基因表現之突變株特性分析 29 1. 泳動能力分析 (swimming motility) 29 2. 氧化逆境測試 (paraquat) 29 3. yeeN 之啟動子在不同突變株中受 paraquat 處理後其表現情形 30 第四章 討論 31 I. DUF28 蛋白質廣泛存在於各物種中，且立體結構十分相似 31 II. yeeN 與 yebC 在調節泳動能力上具有相似的功能 32 III. yeeN 參與大腸桿菌氧化逆境的調控 33 IV. yeeN 可能不參與抗生素逆境之調節 35 V. yebC 可能參與逆境之調節 35 VI. yebC 與 yeeN 是否參與quorum sensing 之調節 36 VII. 參與泳動力及氧化逆境調節之上游基因特性分析 37 VIII. recA 在氧化逆境下可能參於調節 yeeN 基因 38 IX. relA於正常環境中正向調控 yeeN，但在氧化逆境下並無直接參與調節 yeeN 38 X. 在鞭毛生合成過程中 hns 基因可能參與調節 yeeN 39 XI. 結語 40 第五章 未來展望 41 參考文獻 42 | |
| dc.language.iso | zh-TW | |
| dc.title | 大腸桿菌DUF28蛋白質在氧化逆境反應之功能研究 | zh_TW |
| dc.title | Functional study of Escherichia coli DUF28 proteins in oxidative stress response | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳文盛,葉開溫,葉國楨,林乃君 | |
| dc.subject.keyword | 大腸桿菌,DUF28蛋白,yeeN,yebC,氧化逆境,泳動力, | zh_TW |
| dc.subject.keyword | Escherichia coli,DUF28 protein,yeeN,yebC,oxidative stress,motility, | en |
| dc.relation.page | 93 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2011-02-15 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| 顯示於系所單位： | 植物科學研究所 | |
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