絲瓜簇葉病植物菌質體胸

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姓名	江施幸(Shih-Hsing Chiang) 查詢紙本館藏	畢業系所	生命科學系
論文名稱	絲瓜簇葉病植物菌質體胸 (Expression and characterization of the thymidylate kinase from the phytoplasma associated with the loofah witches' broom)
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摘要(中)	本實驗為研究絲瓜簇葉病植物菌質體tmk 基因段。以引子tmk-1與 tmk-2，利用聚合?連鎖反應(PCR) 擴增絲瓜簇葉病植物菌質體tmk 基因段，再利用EcoR1 與 BamH1 限制?切位將此基因段構築至pGEX-2T 蛋白質表現載體之中。將此重組的載體表現在大腸桿菌XL1-blue寄主細胞中大量表現並且在IPTG誘導之下生合成由GST與胸?酸激?(TMK)組成之融合蛋白。此蛋白質為分子量大約50 kD，在應用親和性Glutathione Sepharose 4B管柱萃取純化以及thrombin酵解之後便得到24 kD左右的胸?酸激?蛋白質。　　此胸?酸激?催化磷酸根基團的轉移從ATP轉給dTMP。連續的分光儀分析加上運用丙酮酸磷酸?與乳酸去氫?做偶合反應，用來分析胸?酸激?酵素活性反應以及酵素動力學研究。在酵素活性反應中還原態的NADH被氧化並且在波長340 nm 吸光值發生變化，我們便可以觀察到胸?酸激?的活化催化作用。其反應pH 值約為pH 7 而鎂離子(Mg++)濃度為1.5 ∼ 2.0 mM 而最佳溫度為30 ℃。酵素胸?酸激?的km 與Vmax 值為 0.058 mM 和 1.967 μmol/min 。
摘要(英)	The tmk gene fragments were amplified on the DNA of phytoplasma associated with loofah witches’’ broom by polymerase chain reaction (PCR) using the two primers, tmk-1 and tmk-2, and constructed into the pGEX-2T expression vector at EcoR1 and BamH1 sites. The recombinant vector was expressed in E. coli XL1-blue and a fusion protein composed of glutathione S-transferase (GST) and thymidylate kinase (TMK) was produced in the presence of IPTG. The protein had a molecular weight of about 50 kD. After purified by Glutathione Sepharose 4B column and treated by thrombin, a TMK protein of about 24 kD was obtained. The enzyme TMK catalyzes the transfer of phosphate group from ATP to thymidine monophosphate. A continuous spectrophotometric assay using the pyruvate kinase (PK) and lactate dehydrogenase (LDH) coupling system was employed for the TMK activity assay. The reduced-form NADH was oxidized in the reaction and the absobance at 340 nm was measured. The optimum pH for the reaction was about 7 and Mg++ concentration was 1.5 ∼ 2.0 mM at 30 ℃. The km and Vmax of enzyme TMK was 0.058 mM and 1.967 μmol/min, respectively.
關鍵字(中)	★ 絲瓜簇葉病植物菌質體 ★ 胸	關鍵字(英)	★ tmk ★ GST-TMK fusion protein ★ Km ★ Vmax ★ PK/LDH coupling reaction ★ phytoplasma
論文目次	Abbreviations………………………..……………………….A Chinese Abstract………………………………………………..i Abstract………………………………………………………...ii I. Introduction………………………………………..….……...1 1. The discovery of phytoplasma………………………….1 2. Characteristics of mollicutes……………………………2 3. Classification of thymidylate kinase……………...…….7 4. Physiological role of thymidylate kinase……………….8 5.The objective of study…………………………….…….12 II. Materials and Methods……………………………………13 1. Polymerase chain reactions (PCR)…………….....……13 2. Purification of plasmid DNA……………………..…...14 3. Digestion with restriction enzymes……………………16 4. Preparation of competent cells………………………...17 5.Dephosphorylation……………………………………..18 6.Ligation reaction………………………….……………19 7. Transformation………………………………….……..19 8. Mini scale fusion protein expression……………………20 9. Large scale fusion protein expression……………………21 10. The quantitative of protein by bradford assay….…….22 11. Protein analysis in Tris-glycine SDS-polyacrylamide gel electrophoresis………………23 12. Purification of GST fusion protein………………….27 13. GST Fusion protein digestion by thrombin……………28 14. Thymidylate kinase kinetics………………………….29 III. Results…………………………………………….31 1. Construction of recombinant expression plasmid………31 2. Expression of TMK protein in E. coli …………………31 3. The properties of TMK enzyme activity………………33 4. The kinetic analysis of the enzyme…………………….35 IV. Tables and figures……………………………………36 V. Discussion……………………………………………….59 VI. References………………………………………………65
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指導教授	何國傑	審核日期	2000-7-18
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