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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 莊榮輝(Rong-Huay Juang) | |
dc.contributor.author | Ying-Chen Hsieh | en |
dc.contributor.author | 謝瑩貞 | zh_TW |
dc.date.accessioned | 2021-06-15T05:55:23Z | - |
dc.date.available | 2015-08-20 | |
dc.date.copyright | 2010-08-20 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-17 | |
dc.identifier.citation | 張世宗 (1999) 甘藷塊根 Chaperonin 及 Proteasome 之分離與性質研究
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Singh D.G., Lomako J., Lomako W.M., Whelan W.J., Meyer H.E., Serwe M. and Metzger J.W. (1995) b-Glucosylarginine: a new glucose-protein bond in a self-glucosylating protein from sweet corn. FEBS Lett 376: 61-64. Smith A.M., Denyer K. and Martin C. (1997) The synthesis of the starch granule. Annu Rev Plant Physiol Plant Mol Biol 48: 67-87. Smith A.M., Zeeman S.C. and Smith S.M. (2005) Starch degradation. Annu Rev Plant Biol 56: 73-98. Sonnewald U., Basner A., Greve B. and Steup M. (1995) A second L-type isozyme of potato glucan phosphorylase: cloning, antisense inhibition and expression analysis. Plant molecular biology 27: 567-576. Streb S., Delatte T., Umhang M., Eicke S., Schorderet M., Reinhardt D. and Zeeman S.C. (2008) Starch granule biosynthesis in Arabidopsis is abolished by removal of all debranching enzymes but restored by the subsequent removal of an endoamylase. Plant Cell 20: 3448-3466. Sun Z.T. and Henson C.A. 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(2004a) Recent developments in understanding the regulation of starch metabolism in higher plants. J Exp Bot 55: 2131-2145. Tetlow I.J., Wait R., Lu Z., Akkasaeng R., Bowsher C.G., Esposito S., Kosar-Hashemi B., Morell M.K. and Emes M.J. (2004b) Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions. Plant Cell 16: 694-708. Tickle P., Burrell M.M., Coates S.A., Emes M.J., Tetlow I.J. and Bowsher C.G. (2009) Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm. J Plant Physiol 166: 1465-1478. van de Wal M., D'Hulst C., Vincken J.P., Buleon A., Visser R. and Ball S. (1998) Amylose is synthesized in vitro by extension of and cleavage from amylopectin. J Biol Chem 273: 22232-22240. Vrinten P.L. and Nakamura T. (2000) Wheat granule-bound starch synthase I and II are encoded by separate genes that are expressed in different tissues. Plant Physiol 122: 255-264. Wattebled F., Dong Y., Dumez S., Delvalle D., Planchot V., Berbezy P., Vyas D., Colonna P., Chatterjee M., Ball S. and D'Hulst C. (2005) Mutants of Arabidopsis lacking a chloroplastic isoamylase accumulate phytoglycogen and an abnormal form of amylopectin. Plant Physiol 138: 184-195. Weber A., Servaites J.C., Geiger D.R., Kofler H., Hille D., Groner F., Hebbeker U. and Flugge U.I. (2000) Identification, purification, and molecular cloning of a putative plastidic glucose translocator. Plant Cell 12: 787-802. Weise S.E., Weber A.P. and Sharkey T.D. (2004) Maltose is the major form of carbon exported from the chloroplast at night. Planta 218: 474-482. Yu Y., Mu H.H., Wasserman B.P. and Carman G.M. (2001) Identification of the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase. Plant Physiol 125: 351-359. Zeeman S.C., Kossmann J. and Smith A.M. (2010) Starch: its metabolism, evolution, and biotechnological modification in plants. Annu Rev Plant Biol 61: 209-234. Zeeman S.C., Delatte T., Messerli G., Umhang M., Stettler M., Mettler T., Streb S., Reinhold H. and Kotting O. (2007) Review: Starch breakdown: recent discoveries suggest distinct pathways and novel mechanisms. Functional plant biology 34: 465. Zeeman S.C., Smith S.M. and Smith A.M. (2004a) The breakdown of starch in leaves. New Phytologist 163: 247-261. Zeeman S.C., Thorneycroft D., Schupp N., Chapple A., Weck M., Dunstan H., Haldimann P., Bechtold N., Smith A.M. and Smith S.M. (2004b) Plastidial a-glucan phosphorylase is not required for starch degradation in Arabidopsis leaves but has a role in the tolerance of abiotic stress. Plant physiology 135: 849. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47333 | - |
dc.description.abstract | 澱粉磷解酶 (L-SP) 與D-enzyme都是參與澱粉代謝的重要酵素,一般認為L-SP與澱粉合成較為相關 (Tickle et al., 2009),且L-SP經磷酸化後會改變與澱粉合成相關酵素SBEI與SBEIIb的結合能力,進而影響澱粉的生合成 (Tetlow et al., 2004b);而推測D-enzyme主要是參與澱粉的降解過程 (Zeeman et al., 2010)。此外,研究發現大腸桿菌之D-enzyme (malQ) 與1,4-alpha-D-glucan phosphorylase (malP) 位於同一操作組上 (Goda et al., 1997),說明了D-enzyme與SP可能同時有基因表現並共同作用。張世宗 (1999) 在純化蛋白質過程,發現L-SP高分子量活性色帶HX,後續林怡岑發現D-enzyme也有往高分子量位移的現象。另外,由免疫共沉澱結果推測L-SP與D-enzyme可能有交互作用的現象 (林怡岑, 尚未發表)。
本研究乃以L-SP與D-enzyme的交互作用為主軸,先以GST pull-down assay及Far Western確認L-SP與D-enzyme可互相結合,再利用native/SDS 2D-PAGE與膠體過濾法分析,發現HX之次單元體組成可能為四個L-SP及四個D-enzyme次單元體,整體分子量約686 kDa。此外,比較添加磷酸酶抑制劑或CIAP (calf intestine alkaline phosphatase) 對HX形成之影響,發現磷酸化修飾可促進L-SP與D-enzyme結合。酵素活性分析結果顯示,形成HX蛋白質複合體後,大幅提升了D-enzyme活性,且擴大可利用的受質範圍。推測HX作用模式可能先以D-enzyme與受質反應後,再將產物繼續給L-SP進行磷解反應。進一步在不同生長時期的甘藷塊根中,觀察到L-SP含量與甘藷塊根中澱粉的充實程度成正相關,顯示L-SP可能與澱粉合成途徑較為緊密相關;而D-enzyme則在甘藷塊根剛發芽時,其蛋白質的表現量與酵素活性都增加,顯示D-enzyme可能主要參與澱粉降解的途徑。有趣的是,HX則分別在甘藷塊根快速生長時期,與在甘藷塊根發芽後,都有較多的蛋白質量,推測在澱粉快速消長時,植物透過蛋白質後修飾或其他調控形成HX,協助回收可利用的葡聚醣,以利澱粉代謝的進行。 | zh_TW |
dc.description.abstract | Starch phosphorylase (L-SP) and D-enzyme are two important enzymes in starch metabolism. Several groups showed that L-SP is involved in starch biosynthesis (Tickle et al., 2009). L-SP can be phosphorylated, and the phosphorylated L-SP may form protein complexes with SBEI and SBEIIb which then affect the biosynthesis of starch (Tetlow et al., 2004b). On the other hand, it is believed that D-enzyme is mainly participated in starch degradation (Zeeman et al., 2010). Besides, it was found that D-enzyme (malQ) and 1, 4-alpha-D-glucan phosphorylase (malP) are encoded by the genes of the same operon in E. coli (Goda et al., 1997), suggesting that D-enzyme and SP may work together. During the purification process of L-SP, Chang (1999) found a high-molecular weight band (HX) showing L-SP activity. Subsequently, Lin also found that D-enzyme showed an extra form of catalytic activity having high molecular weight during its purification. The interaction of D-enzyme and L-SP was confirmed by co-immunoprecipitation (Lin, unpublished observation).
In this study, GST pull-down assay and Far Western were utilized to confirm the protein-protein interaction between L-SP and D-enzyme. It was found that HX may be composed of four L-SP and four D-enzyme subunits, and the molecular weight of HX was estimated as 686 kDa by gel filtration and native/SDS 2D-PAGE. In addition, we checked the effect of phosphorylation/dephosphorylation on the formation of HX, and found that post-translational phosphorylation may promote the interaction of L-SP and D-enzyme. Moreover, high molecular weight form (HX) of D-enzyme demonstrated an enhanced catalytic activity, and showed wider range for various substrates. Therefore, in the protein complex (HX), D-enzyme might play the role in producing the malto-oligosaccharides which were then acted by L-SP via phosphorolysis. Furthermore, the protein level of L-SP increased when sweet potato roots accumulating starch, but decreased when sweet potato roots sprouted. This result showed that L-SP might be closely related to starch biosynthesis. However, the enzyme activity and protein expression of D-enzyme increased when sweet potato roots was about to sprout, indicating that D-enzyme might be mainly involved in starch degradation. Interestingly, the protein level of HX increased at two physiological stages: firstly, when the starch synthesis was active; and secondly, after sweet potato roots were germinated. These results suggested that the formation of HX might go through post-translational modification, which may then facilitate the starch metabolism by recycling the available glucans. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T05:55:23Z (GMT). No. of bitstreams: 1 ntu-99-R97b47203-1.pdf: 3392026 bytes, checksum: 71d283f7909fc2903a39630d0dbf6342 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 目 錄 I
中文摘要 III Abstract IV 第一章 緒 論 1 1.1 植物澱粉生合成的主要途徑 2 1.1.1 adenosine-5’-diphosphate glucose (ADPGlc) 合成 2 1.1.2 葡聚醣鏈延長 3 1.1.3 澱粉的分支 4 1.2 澱粉合成之酵素形成蛋白質複合體 7 1.3 合成澱粉粒的起始反應與調控 8 1.4 澱粉降解途徑 10 1.4.1 葉綠體中的澱粉降解途徑 10 1.4.2 葉片及穀物胚乳中澱粉降解途徑之差異 12 1.5 澱粉磷解酶 15 1.5.1 澱粉磷解酶與其他蛋白質交互作用與磷酸化之影響 18 1.6 D-enzyme 19 1.6.1 D-enzyme與L-SP之關聯性 21 1.7 研究動機 23 第二章 材料與方法 25 2.1 實驗材料 25 2.1.1 植物材料 25 2.1.2 表現載體 (vectors) 25 2.2 實驗藥品 25 2.3 儀器設備 26 2.4 實驗方法 28 2.4.1 添加磷酸酶抑制劑與蛋白質去磷酸化分析 28 2.4.2 Far Western assay 28 2.4.3 Pull-down assay 30 2.4.4 native/SDS 2D-PAGE 32 2.4.5 FPLC型膠體過濾法 32 2.4.6 酵素活性分析 32 2.4.7 甘藷塊根中相異蛋白質之含量分析 34 第三章 結果與討論 35 3.1 L-SP之蛋白質交互作用 35 3.1.1 以甘藷塊根粗抽液進行Far Western 35 3.1.2 處理NaF, Na3VO4 及CIAP對HX形成之影響 38 3.1.3 HX與D-enzyme之GST pull-down assay 40 3.2 蛋白質複合體組成之分析 42 3.2.1 以native/SDS 2D-PAGE觀察HX之組成 42 3.2.2 以膠體過濾法推測HX之分子量 45 3.2.3 分析組成HX之次單元體數 47 3.3 藉由酵素活性分析推測HX的作用模式 48 3.4 觀察HX、L-SP及D-enzyme在不同生長時期可能的生理角色 54 第四章 總結 57 參考文獻 59 附錄 63 附錄1一般分析法 63 附錄1.1 蛋白質定量法 63 附錄1.2 甘藷塊根L-SP的磷酸活性分析法 64 附錄1.3 甘藷塊根D-enzyme的活性分析方法 65 附錄1.4 甘藷塊根L-SP的活性分析方法 (磷解方向) 67 附錄2一般電泳檢定法 69 附錄2.1 原態膠體電泳 69 附錄2.2 SDS膠體電泳 72 附錄2.3 Native/SDS 2D-PAGE膠體電泳 74 附錄2.4膠體染色法: 75 附錄2.5 膠片乾燥法及護貝 82 附錄2.6蛋白質電泳轉印法 83 附錄3免疫學方法 85 附錄3.1 酵素免疫染色法 85 附錄4甘藷塊根L-SP粗抽取法 87 附錄4.1 酵素粗抽取及硫酸銨分劃 87 附錄4.2 管柱色層分析法之操作 89 附錄4.3 離子交換法 91 附錄4.4 膠體過濾法 92 附錄4.5 疏水性層析法 93 附錄5甘藷塊根L-SP高分子量色帶HX純化法 94 附錄6甘藷塊根L-SP110純化法 96 附錄7大腸桿菌表現蛋白質之誘導與純化 98 附錄8 Far Western 101 附錄9 GST pull-down assay 102 | |
dc.language.iso | zh-TW | |
dc.title | 澱粉磷解酶與D-酵素的交互作用與在植物代謝的可能角色 | zh_TW |
dc.title | The interaction of starch phosphorylase and D-enzyme and its
possible role in plant metabolism | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳翰民,王金和,常怡雍,張世宗 | |
dc.subject.keyword | 澱粉磷解酶,蛋白質交互作用, | zh_TW |
dc.subject.keyword | Starch phosphorylase,D-enzyme,Protein-protein interaction, | en |
dc.relation.page | 102 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-18 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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