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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 謝旭亮(Hsu-Liang Hsieh) | |
dc.contributor.author | Wei Siao | en |
dc.contributor.author | 蕭蔚 | zh_TW |
dc.date.accessioned | 2021-06-17T00:01:40Z | - |
dc.date.available | 2012-07-27 | |
dc.date.copyright | 2012-07-27 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-07-16 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65724 | - |
dc.description.abstract | 植物會持續偵測生長的環境並隨之改變他們的生理代謝、生長及發育,因此外界不同波長的光及植物體內各式荷爾蒙,對於決定植物個體的型態都扮演著很重要的因素。在幼苗發育時期,已知FAR-RED INSENSITIVE 219 (FIN219) 會參與在光敏素A及茉莉酸訊息傳遞路徑中,影響植物光型態形成及茉莉酸反應。但是FIN219蛋白質之轉譯後修飾及其調控機制仍未研究釐清。透由電腦軟體預測顯示FIN219有酪蛋白激酶 (Casein Kinase 2) 之磷酸化位點,並利用試管內磷酸激酶分析試驗證實酪蛋白激酶可磷酸化FIN219,其位點為蘇氨酸 (threonine) 55及69。此外,未磷酸化之FIN219在合成茉莉酯-L-異亮胺酸 (Jasmonyl-L-isoluecine) 之反應擁有較低的解離常數Kd值,代表其對受質有較高的結合性;此外,去磷酸化後之FIN219其穩定性則較差易被分解。我們也發現FIN219在植物體內存在兩種型式,且質量低的型式會因酪蛋白激酶抑制劑4,5,6,7-Tetrabromobenzotriazole (TBB)處理後增加,而且其兩種型式之比例會被遠紅光處理所調節改變。外表型檢測顯示fin219-2對於TBB抑制下胚軸及根長之現象皆較不敏感,並且此抑制下胚生長之現象會被不同強度之遠紅光影響。整合以上,我們提出第一份酪蛋白激酶參與在茉莉酸訊息傳遞路徑中的證據,並且說明FIN219磷酸化狀態之變化參與調節遠紅光及茉莉酸的訊息溝通。 | zh_TW |
dc.description.abstract | Plants continuously monitor their surrounding environments and change accordingly their physiological metabolism, growth and development. Different spectrums of light and various hormones are essential to determine the shapes and morphologies of individual plants. During the seedling stage, FAR-RED INSENSITIVE 219 (FIN219) has been shown to participate in phytochrome A and Jasmonate signaling pathways, exerting its functions in photomorphogenesis and JA responses. However, the effects of the posttranslational modifications and related regulatory mechanisms on FIN219 protein levels remain to be elucidated. In silico study shows that FIN219 is predicted to contain casein kinase 2 (CK2) phosphorylation sites, and in vitro kinase assay reveals that FIN219 can be indeed phosphorylated by CK2 on the sites of threonine 55 and threonine 69. Moreover, the unphosphorylated form of FIN219 exhibits lower Kd toward the conjugating process of JA-Isoleucine formation, which means higher affinity to its substrates, and is prone to undergo degradation events. We also observed two FIN219 isoforms in planta whose ratios are modulated by 4,5,6,7-Tetrabromobenzotriazole (TBB), a CK2 inhibitor, and by FR light treatments. Phenotypic examinations show that fin219-2 is less sensitive to TBB inhibition on both hypocotyl and root elongation, and the TBB inhibition of hypocotyl growth are modulated by different intensities of far-red light. Taken together, our study provides the first evidence that CK2-mediated phosphorylation of FIN219 participates in JA signaling during seedling development, and is further involved in the cross talk between JA and FR light signaling pathways. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T00:01:40Z (GMT). No. of bitstreams: 1 ntu-101-R99b42022-1.pdf: 2488491 bytes, checksum: 26b30ee7c31ee6bf23fc072a1ac59db2 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 誌謝 I
中文摘要 II Abstract III Abbreviation V Table of Contents VI Introduction 1 Light perception in plants 1 Phytochrome signaling pathways 1 General structures of phytochrome A 3 The kinase activity of phytochrome A 3 The physiological functions of phytochrome A dependent phosphorylation 4 Phytochrome A regulates the nuclear abundance of COP1 5 FIN219/JAR1 is a dual function protein participating in FR and JA signaling pathways 6 Casein kinase 2 phosphorylation regulates photomorphogenesis in Arabidopsis 7 Materials and methods 9 Plant Materials 9 Gene Constructions 9 GST-tag Protein Purification 9 In vitro CK2 Kinase assay 10 Pro-Q Diamond Staining 10 Plant Growth Conditions 10 Molecular Graphics and Protein Visualization 11 Plant Total Protein Extraction 12 Western Blot Analysis 12 Measurement of Hypocotyl Length 12 Measurement of Primary Root Length 13 Microscopy 13 Results 14 FIN219 is predicted to contain CK2 phosphorylation sites. 14 CK2 phosphorylates FIN219 at Threonine 55 and Threonine 69 in vitro. 15 Mapping of CK2 phosphorylation sites in FIN219/JAR1 3D structure 16 FIN219 has phospho-isoforms in vivo that are regulated by far-red light. 17 Unphosphorylated FIN219/JAR1 has higher affinity to its substrate. 19 TBB inhibits the root elongation in a FIN219 dependent manner. 20 TBB inhibits root hair growth and diminishes the level of FIN219 in root hairs. 20 Phosphorylated FIN219 is functionally dominant in strong cFR response 21 Discussion 24 Phosphorylation of FIN219 protein by casein kinase 2 24 Separation of FIN219 isoforms by western blots 26 Phosphorylation of FIN219 fine-tunes the balance between far-red light and jasmonate signaling. 28 Figures and Tables 32 Table 1. In Silico Prediction of CK2 phosphorylation sites in FIN219. 32 Figure 1. CK2 phosphorylates FIN219 within the region of N45 to N70. 33 Figure 2. FIN219 is phosphorylated by CK2 at threonine 55 and threonine 69. 34 Figure 3. CK2 phosphorylation sites in FIN219/JAR1 protein. 35 Figure 4. FIN219 has phospho-isoforms in vivo. 36 Figure 5. The protein levels and the ratio of phosphorylated/unphosphorylated FIN219 are modulated by continuous far-red light in vivo. 37 Figure 6. Unphosphorylated FIN219 has higher affinity to its substrate. 38 Figure 7. TBB inhibits the root elongation in a FIN219 dependent manner. 39 Figure 8. TBB inhibits root hair growth and diminish the level of GFP-FIN219 in root hairs. 40 Figure 9. FIN219 is involved in the TBB inhibition of hypocotyl growth and the far-red light inhibitory effect is FIN219 phosphorylation dependent in strong cFR. 41 Figure 10. CK2-mediated phosphorylation of FIN219 fine-tunes the balance between FR responses and JA responses. 42 Supplementary Data 43 Figure S1. Red light or far-red light pulses do not affect the phosphorylation states of FIN219 obviously. 43 Figure S2. MeJA elevates the FIN219 protein levels both in the dark and continuous far-red light condition. 43 Figure S3. Capillary isoelectric focusing electrophoresis revealed that FIN219 has three isoforms with different isoelectric points. 44 Figure S4. PhosTag acrylamide gel electrophoresis is not able to separate the FIN219 isoforms clearly. 45 Figure S5. Laemmli SDS-PAGE gel electrophoresis showed varied patterns of FIN219 isoforms. 45 Table S1. The western blot conditions suitable for separating FIN219 isoforms. 46 Figure S6. Strong far-red light sometimes shifts FIN219 to the lower isoforms without diminishing the protein levels, while TBB treatment sometimes leads to FIN219 degradation. 46 Reference 47 Appendix 53 Detailed Protocol 53 Buffer Formula 58 Primer List 60 | |
dc.language.iso | en | |
dc.title | FIN219/JAR1藉由酪蛋白激酶2之磷酸化調控遠紅光及茉莉酸訊息溝通路徑 | zh_TW |
dc.title | CK-mediated Phosphorylation of FIN219/JAR1 Fine-tunes the Cross-talk between Far-red Light and Jasmonate Signaling Pathway | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 符宏勇(Hong-Yong Fu),王淑珍(Shu-Jen Wang),涂世隆(Shih-Long Tu),陳建德(Chien-Teh Chen) | |
dc.subject.keyword | 酪蛋白激酶,磷酸化,遠紅光,茉莉酸, | zh_TW |
dc.subject.keyword | FIN219,JAR1,CK2,Phosphorylation,Far-red light,Jasmonate, | en |
dc.relation.page | 61 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-07-16 | |
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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