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學校名稱輔仁大學
系所名稱生命科學系
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學號493546132
研究生(中)張雅傑
研究生(英)Ya-Chieh Chang
論文名稱(中)光合菌FJ1 phbP基因的表現受PHB含量所影響
論文名稱(英)The Effect of Polyhydroxybutyrate on Expression of phbP in Rhodobacter sphaeroides FJ1
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指導教授(中)楊美桂
指導教授(英)Mei-Kwei Yang
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關鍵字(中)聚3-羥基丁酯 聚羥基烷酯類 光合菌 phbP基因 冷光酶 啟動子活性
關鍵字(英)PHB PHA R. sphaeroides FJ1 phbP luciferase promoter activity
摘要(中)光合菌R. sphaeroides FJ1之phbP基因,座落於phbC基因下游153 bp處,由450 bp構成。分別取含有phbP基因轉譯起始碼ATG上游216 bp,91 bp及41 bp序列之不同片段,構築三個phbP – luxAB 融合之重組質體,送入大腸桿菌Escherichia coli JM109。含pHPP216及pHPP91質體之轉形菌皆具有冷光?活性,表示phbP基因具有獨立之啟動子,且其有效之啟動子範圍為ATG上游91個核?酸內。光合菌FJ1之phbP基因,可轉譯出150個胺基酸,分子量為17 kDa之PhbP 蛋白。構築含重組質體pHPE45之轉形菌後,表現並純化17 kDa PhbP蛋白後,製備抗PhbP蛋白之抗體。以抗PhbP蛋白之抗體進行西方墨點反應,測定PhbP蛋白之合成。欲知PHB含量對phbP基因表現之影響,以碳氮莫耳數比值為20與40之二次培養基增加PHB產量,並分析phbP之表現。發現phbP之表現量會隨PHB含量增加,故PHB含量會影響phbP表現。欲知phbR基因對phbP表現之影響,比較phbP在FJ1野生株與phbR基因突變株中之表現。phbR 突變株之PhbP蛋白大量合成,表示PhbR蛋白可抑制phbP之表現。PHB含量多時,PhbP蛋白之合成亦較多,故PhbR蛋白抑制phbP基因表現較不明顯。PHB含量少時,PhbP蛋白之合成亦較少,故PhbR蛋白抑制phbP基因表現較明顯。因此PHB含量會影響PhbR蛋白抑制phbP基因之程度,故phbP基因之表現會受PHB及phbR影響。
摘要(英)phbP gene was identified in the polyhydroxybutyrate synthetic locus ( phbZ-C-P-R ) of Rhodobacter sphaeroides FJ1. phbP of R. sphaeroides FJ1 located in downstream 153 base pairs of phbC, is consist of 450 bp.To analyse whether phbP has its independent promoter, three fragments upstream 216 bp, 91 bp, and 41 bp of phbP transltaion start site were cloned into pMY1 to generate pHPP216, pHPP91, and pHPP41 plasmid. The luciferase activity of transformants carrying pHPP216 and pHPP91 plasmid is 5.12 and 4.33 RLU(×106). So phbP has its self promoter and its active promoter range is among upstream 91 bp of ATG . phbP of R. sphaeroides FJ1 has a open reading frame that can encodes 149-amino acid protein, and moleculor weight of its protein is 17 kDa. After 17 kDa PhbP in Escherichia coli carrying a pHPE45 plasmid was expressed and purified, preparing anti-PhbP antibody. Expression of phbP in R. sphaeroides FJ1 was determined by western immunoblotting. Utilizing fed-batch methods, accumulation of PHB can increases expression of phbP when FJ1 was grown in medium containing excess carbon and limited nitrogen. phbP overexpresses in phbR mutant strain, so expression of phbP could be inhibited by PhbR .When content of PHB increases, PhbR decreasely inhibits phbP express. When content of PHB decreases, PhbR apperantly inhibits phbP express. These results indicated expression of phbP in R. sphaeroides FJ1 would be affected by both PHB and phbR .
論文目次目錄 I 圖目錄 0III 中文摘要 0IV Abstract V 前言 001 材料與方法 10 一、質體 010 二、藥品、酵素及耗材 012 三、培養基 013 四、緩衝溶液與試劑 014 五、實驗方法 190 1.菌體之培養及保存 015 2. DNA抽取 020 3.聚合?鏈鎖反應 021 4.重組質體之選殖 022 5.洋菜膠電泳分析 025 6. DNA之定序及序列比對 026 7.蛋白質之純化 026 8. SDS-聚丙烯醯胺凝膠電泳分析 028 9.製備抗PhbP蛋白之抗體 029 10.西方墨點法 030 11.PHA產量測定 031 12.冷光?活性分析 031 結果 032 一、phb基因之啟動子分析 32 (一)phbP基因之啟動子分析 0 32 1.質體pHPP216,pHPP91與pHPP41之構築 032 2. phbP啟動子之活性及有效啟動範圍確定 33 (一)phbZ基因之啟動子分析 0 36 1.質體pHZP115與pHZP51之構築 036 2. phbZ啟動子之活性及有效啟動範圍確定 37 二、phbP基因之表現 40 (一)光合菌FJ1 PhbP蛋白之純化 40 1.重組質體pHPE45之構築 40 2. PhbP蛋白之純化 0 41 (二)phbP基因之表現受PHB含量所影響 44 1. 培養於TSB培養基PhbP蛋白之合成 44 2. 培養於高碳氮比例培養基PhbP蛋白之合成 46 三、phbP基因之表現受PhbR蛋白所影響 48 討論 043 參考資料 48 圖 目 錄 圖1. 重組質體pHPP216,pHPP91及pHPP41之構築 034 圖2. 光合菌 FJ1 phbP基因啟動子之活性分析 035 圖3. 重組質體pHZP115及pHZP51之構築 038 圖4. 光合菌 FJ1 phbZ基因啟動子之活性分析 039 圖5. 光合菌FJ1 phbP基因重組質體 pHPE45之構築 42 圖6. phbP基因表現之誘導與PhbP蛋白之純化 43 圖7. 光合菌FJ1培養於TSB培養基PhbP蛋白之合成 45 圖8. 光合菌FJ1培養於高碳氮比例培養基PhbP蛋白之合成 47 圖9.比較光合菌FJ1野生株與phbR突變株PhbP蛋白之合成 049
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