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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張英峯(Ing-Feng Chang) | |
| dc.contributor.author | Man-Hsuan Lee | en |
| dc.contributor.author | 李曼瑄 | zh_TW |
| dc.date.accessioned | 2021-06-08T03:49:20Z | - |
| dc.date.copyright | 2021-01-07 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-12-28 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21843 | - |
| dc.description.abstract | 乙烯是調節植物發育過程的重要植物荷爾蒙之一。1-氨基環丙烷-1-羧化合成酶（ACS）蛋白質在乙烯生合成途徑扮演關鍵角色。第三型ACS7具有非常短的C端延伸，缺乏鈣離子依賴性蛋白質激酶（CDPK）和絲裂原活化蛋白質激酶6 （MPK6）可被磷酸化之位點，但之前研究發現ACS7可以被CDPK16磷酸化，並鑑定出三個磷酸化位點。然而目前仍未知ACS7的三個磷酸化位點在蛋白穩定度及活性的功能。另一方面，先前的研究發現阿拉伯芥acs7-1突變體有較高的耐鹽性，且在我們先前的研究顯示acs7-1突變體在根向地性對鈣離子通道阻斷劑氯化鋰不敏感。為了探討ACS7的三個磷酸化位點在根向地性及耐鹽性的功能，本研究以油菜內酯、離層酸、鹽及氯化鋰或鈣離子螯合劑BAPTA進行處理。acs7-1突變體幼苗在向地性顯示對氯化鋰、離層酸及BAPTA較不敏感，但對油菜內酯較敏感。此外，ACS7的三個磷酸化位點之點突變轉殖株對鹽分之耐受性及氯化鋰處理下之根向地性抑制程度與未突變之轉殖株有別。另外，似乎在Ser216, Thr296, 和Ser299點突變之ACS7-GUS轉殖株中ACS7蛋白質穩定性較未突變之轉殖株差，且ACS7的三個磷酸化位點突變之ACS7重組蛋白質比未突變者酵素活性來得低，因此，本研究發現ACS7的三個磷酸化位點會影響ACS7蛋白質的活性且似乎也會影響蛋白穩定性。 | zh_TW |
| dc.description.abstract | Ethylene is one of the important plant hormones that regulate the developmental processes in plants. 1-aminocyclopropane-1-carboxylase synthase (ACS) proteins play important role in the ethylene biosynthesis pathway. Type III protein ACS7 has a very short C-terminal extension that lacks both predicted calcium-dependent protein kinase (CDPK) and mitogen-activated protein kinase 6 (MPK6) phosphorylation sites. Our previous study showed that ACS7 protein can be phosphorylated by CDPK16 in vitro, and identified three phosphorylation sites. However, the function of the three phosphorylation sites in ACS7 protein stability and activity are still unknown. On the other hand, previous studies showed acs7-1 mutant had a higher salt tolerance in Arabidopsis. acs7-1 mutant showed insensitive to lithum chloride, a calcium channel blocker, in root gravitropism in our previous study. In order to investigate the function of the three phosphorylation sites of ACS7 in root gravitropism and salt tolerance, brassinolide (BL), abscisic acid (ABA), salt, and LiCl or a calcium chelator, 1,2-bis (o-aminophenoxy) ethane-N,N,N’ ,N’ -tetraacetic acid (BAPTA) were introduced in this study. In the acs7-1 mutant, the seedlings showed less sensitive to LiCl, ABA, and BAPTA, but it’s more sensitive to BL in root gravitropism. In addition, the three phosphorylation site Ser216, Thr296, and Ser299 mutated ACS7-β-Glucuronidase (GUS) transgenic lines exhibited differential salt tolerance and root gravitropism in comparison to non-mutated transgenic lines. On the other hand, ACS7 phosphorylation site Ser216, Thr296, and Ser299 point mutated GUS transgenic lines appear to show less protein stability compared to none mutated line. Moreover, ACS7 phosphorylation site point mutated recombinant protein exhibited lower ACS7 enzyme activity than none mutated one. Taken together, the three phosphorylation sites of ACS7 can affect protein activity and appear to affect protein stability of ACS7. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T03:49:20Z (GMT). No. of bitstreams: 1 U0001-2412202015371300.pdf: 4790462 bytes, checksum: 4681c2a6413e11578ddae6782f8df073 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 致謝…………………………………………………………………………………….I 摘要................................................................II Abstract............................................................III Contents.............................................................V List of Table………………………………………………………………………..X List of Figures……………………………………………………………………..XI List of Supplementary data…………………………………………………………XIII List of Appendixes……………………………………………………………...….XIV Abbreviation………………………………………………………………………..XVI 1. Introduction……………………………………………………………………...1 1.1 Ethylene biosynthesis pathway……………………………………………….1 1.2 ACC synthase and the regulation on protein stability………………..2 1.3 14-3-3 proteins………………………………………………………………...4 1.4 Calcium signaling……………………………………………………………...5 1.5 Calcium-dependent protein kinase (CDPK)……………….....……………6 1.5.1 The Arabidopsis CDPK gene family………………………………........6 1.5.2 Functional domains of CDPKs…………………………………….........6 1.6 Root gravity response………………………………………………………...7 1.7 Plant hormone auxin in root gravity response…………………………..8 1.8 Aux/IAA-based reporter, domain II (DII)-VENUS………………………….9 1.9 Plant hormone brassinosteroids in root gravity response……………10 1.10 Plant hormone abscisic acid……………………………………………….11 1.11 Salt stress responses in plants………………………………………….12 1.12 Ethylene signaling in salt-stress tolerance in plants…………...12 1.13 Project goals………………………………………………………………...13 2. Materials and Methods………………………………………………………….16 2.1 Plant materials and plant growth conditions…………………………..16 2.2 Escherichia coli transformation…………………………………………..16 2.3 Agrobacterium tumefaciens transformation……………………………….17 2.4 Floral dipping………………………………………………………………...17 2.5 Purification of glutathione S-transferase (GST)-tagged proteins..18 2.6 Purification of 6His-SUMO-ACS7 recombinant proteins…………………19 2.7 Protein quantification by Bradford assay……………………………….20 2.8 Isolation of Arabidopsis leaf protoplasts for bimolecular fluorescence complementation (BiFC) assay………………………………………….…......20 2.9 Transformation of plasmids for BiFC analysis in Arabidopsis protoplasts…..21 2.10 In vitro kinase assay………………………………………………………...........22 2.11 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot…………………………………………………........................22 2.12 RNA extraction and gene expression analysis……………………….............24 2.13 Semi-quantitative and quantitative real-time PCR analysis……………….....24 2.14 Root curvature and the ratio of gravity sustaining measurement………......25 2.15 Primary root length measurement…………………………………………...........25 2.16 Seed germination rate and floral dipping measurement…………………........26 2.17 in vitro ACS7 enzyme activity assay……………………………………….........26 2.18 Phosphoproteome analysis……………………………………………................27 2.19 DII-VENUS fluorescence observation in roots…………………………...........30 2.20 Histochemical β-Glucuronidase (GUS) staining………………………...........30 2.21 Protein degradation assay……………………………………………….............31 2.22 Cell-free protein degradation assay……………………………………….........31 3 Results…………………………………………………………………………..............33 3.1 Isolation of T-DNA insertional mutant acs7-1 and generation of ACS7 transgenic lines…………………………………………………………….................33 3.2 Observation of DII-VENUS fluorescence in roots against gravity under multiple drug-treated conditions…………………………………………….............34 3.3 Gravity response and root curvature of acs7-1 mutant line against gravity under BAPTA-treated condition……………………………………………………................35 3.4 Gravity response of ACS7 transgenic lines and acs7-1 mutant line under BL- treated condition……………………………………………………………................36 3.5 Primary root growth and root curvature of acs7-1 mutant line in ABA response……………………………………………………………………...................37 3.6 Root curvature of ACS7 transgenic lines and acs7-1 mutant line against gravity under LiCl-treated condition………………………………………………...............37 3.7 Survival percentage of ACS7 transgenic lines and acs7-1 mutant line under salt stress condition…………………………………………………………….................39 3.8 In vitro enzyme activity assay of three phosphorylation site mutated variants of ACS7 recombinant proteins…………………………………………………................39 3.9 Expression pattern of ACS7-GUS lines in multiple treatment conditions…....40 3.10 Protein degradation assay of ACS7-GUS lines……………………….............41 4 Discussion……………………………………………………………………….............43 4.1 The function of the three phosphorylation sites of ACS7 in stability and activity……………………………………………………………………….................43 4.2 The function of the three phosphorylation sites of ACS7 in survival percentage.....................................................................45 4.3 The relationship between ACS7 and ABA………………………………..............45 4.4 Phenotype of acs7-1 in BAPTA treatment in root gravitropism……………......46 4.5 Phenotype of acs7-1 in BL treatment in root gravitropism……………….......46 4.6 Fluorescence analysis of DII-VENUS under different treatment conditions....47 References…………………………………………………………………….................49 Table……………………………………………………………………………................69 Figures…………………………………………………………………………................70 Supplementary data……………………………………………………………...............89 Appendixes……………………………………………………………………................101 | |
| dc.language.iso | zh-TW | |
| dc.title | ACS7磷酸化在阿拉伯芥根向地性與鹽耐受性之功能性研究 | zh_TW |
| dc.title | Functional study of ACS7 phosphorylation in root gravitropism and salt tolerance in Arabidopsis thaliana | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 109-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 靳宗洛(Tsung-Luo Jinn), 鄭秋萍(Chiu-Ping Cheng), 李金美(Chin-Mei Lee),吳俊達(Chun-Ta Wu) | |
| dc.subject.keyword | 乙烯,ACS7,磷酸化,鹽,離層酸,活性,穩定度, | zh_TW |
| dc.subject.keyword | Ethylene,ACS7,phosphorylation,salt,ABA,activity,stability, | en |
| dc.relation.page | 112 | |
| dc.identifier.doi | 10.6342/NTU202004456 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-12-29 | |
| 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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