大豆是世界上最重要的經濟作物之一。然而，栽培大豆的絶大部分區域等均不是肥沃土壤。據Boyer 估計，將近70%的大豆產量潛能因為立地條件不適宜而不能被挖掘出來，哪怕是在農業發達地區也是如此。因此，本課題立足於探究大豆耐鹽抗性機理的研究，籍此為耐鹽性大豆品種的開發提供科學依據。 / 蛋白質免疫印記方法（Western blotting）被運用于對已知的大豆組蛋白H3、H4的轉錄後修飾中的全部磷酸化、甲基化和乙酰化修飾標記進行掃描式測試，以分析鹽脅迫情況下這些表觀遺傳標記的動態變化情況。結果表明，組蛋白H3的Lysine 9 和Lysine14 乙酰化（H3K9Ac，H3K14Ac）以及Serine 10 磷酸化（H3Ser10Phos）在鹽處理前後變化顯著。該結果預示著這些表觀遺傳標記與大豆耐鹽反應之間可能存在著密切的關聯。因此，後續工作將圍繞它們展開。 / 染色體免疫共沈澱的技術（ChIP）被應用于分析與組蛋白標記H3K9Ac，H3K14Ac 和H3Ser10Phos 等相互作用的細胞核內因子，包括核內蛋白質和gene等。通過SDS-PAGE 的MALDI-TOF/ TOF 發現，與H3Ser10Phos 相互作用的因子中有含PHD 蛋白域的核內蛋白。我們以前的試驗結果曾揭示PHD 蛋白能夠招募一個能夠專一地乙酰化組蛋白標記H3K14 的乙酰化酶GNAT，顯然，該結果預示著H3Ser10 的磷酸化與H3K14 的乙酰化之間有著內在的聯系並依次順序發生。進一步的試驗表明，該蛋白域能夠招募大豆ISWI 和Elongin A 蛋白。這兩個蛋白可以分別重塑染色體空間結構和促使轉錄延伸功能。同時，H3K14Ac 可以招募翻譯延長因子Translation Elongation Factor Ts。 / 為進一步以蛋白組學策略了解H3K9Ac，H3K14Ac 和H3Ser10Phos 等表觀遺傳標記協同調節耐鹽基因表達活性的機理，我合成了帶這些修飾標記的八條肽做Pull down 試驗。這八條肽包含了一條沒有修飾的肽（H3）作為對照以及單個位點修飾（ 如H3K9Ac ， H3K14Ac ， H3Ser10Phos ）、雙位點修飾（ 如H3K9AcSer10Phos ， H3K9AcK14Ac 、H3Ser10PhosK14Ac ） 和三位點修飾（H3K9AcSer10PhosK14Ac）。結果發現，所有修飾肽段都能招募到rubber elongation factor protein 和peroxidase 1 precursor 蛋白。同时，所有含K14Ac 標記的肽（如H3K14Ac、H3Ser10PhosK14Ac 等）均能與其它組蛋白（如H2B，H4）相互作用。可見，H3K14Ac 是不同組蛋白之間相互“交流“的橋梁。此外，14-3-3 以及GmAPX 等均被發現與H3Ser10Phos 相互作用，進一步印證了前人預言的抗壞血酸-谷胱甘肽循環系統中的某些酶可能存在反饋調節機制的構想，以維持系統內氧化還原狀態的平衡。 / 爲了解上述組蛋白標記與耐鹽基因相互作用的結合位點，我們運用了染色體免疫共沈澱方法。結果表明，H3K14Ac 可以與大豆耐鹽相關基因GmGST（編碼谷胱甘肽-S-轉移酶蛋白）啟動子區結合。我們同時又運用相同方法測試了H3K14 鄰近兩個組蛋白標記H3K9Ac 和H3Ser10Phos，結果發現H3K9Ac 亦可以與該基因的啓動子區域結合，而H3Ser10Phos 可能直接結合與該基因的編碼區。上述試驗表明，H3K9Ac，H3K14Ac 和H3Ser10Phos 等標記有調節耐鹽基因GmGST 表達水平的潛能。 / 通過蛋白組學方法發現，GmGST 編碼的谷胱甘肽S-轉移酶在鹽處理後蛋白質含量顯著升高。而且，以谷胱甘肽S-轉移酶為關鍵酶的抗壞血酸-谷胱甘肽循環系統中的其它幾個酶，如GmMDAR 和GmAPX 等，亦顯著升高。顯然，抗壞血酸-谷胱甘肽循環系統中的上述酶在清除鹽脅迫條件下劇增的過氧化物（如H₂O₂）有直接作用，進而保護大豆免受鹽脅迫的危害。 / 綜合上述結果，我們提出一個假說來闡述組蛋白H3 的N-末端PTMs，GmGST和GmAPX 等核內蛋白因子協同作用調節大豆耐鹽能力的機理，以適應鹽脅迫逆境中大豆植株積累起來的過氧化物危害。最初，細胞內活性氧物的積累（如過氧化氫），會引起H3S10Phos 和H3K14Ac 等組蛋白修飾，隨之這些修飾可能促使耐鹽基因GmGST 的表達，然後增加胞漿GmGST 蛋白含量，從而直接或間接地調節谷胱甘肽抗壞血酸循環中下遊的GmMDAR 和GmAPX 酶表達量。由于GmAPX 具有抗氧化作用，消除過量積累的過氧化氫，最終實現大豆耐鹽功能。另一方面，過度積累的GmAPX 又可以返回細胞核內，並反饋給核內調節因子，從而抑制GmGST 基因的過量表達。 / Soybean (Glycine max (L.) Merrill) represents one of the most significant economical legume crops. However, conditions in almost all cultivated land are sub-optimal for plant growth. Boyer estimated that about 70% of the potential yield is lost as a result of unfavorable physiochemical environments, even in developed agricultural systems. This project tried to understand the defensive mechanisms/ responses of soybean upon the salinity stress for further improving its salinity tolerance. / The abundance dynamics of all available histone PTMs have been screened inside the soybean plants under salinity stress and demonstrated that in comparing with the control sample, H3 lysine acetylation (Lys Ac), H3 lysine 14 acetylation (H3K14Ac), H3 Serine 10 phosphorylation (H3S10Phos) increased under the salinity stress. Indicating that H3K14Ac and H3S10Phos might play important roles in soybean adaptation to salinity stress and they were chosen for exploring the mechanisms of soybean tolerance. / To investigate the nucleic factors (Proteins and genes) which interacted with the H3K14Ac, H3K9Ac and H3S10Phos, the precipitated proteins obtained from ChIP assay were further separated by SDS-PAGE and identified by MALDI-TOF/ TOF. Different proteins could be identified among all three PTMs. Results showed that H3S10Phos could recruit PHD finger family protein, which was previously found to interact with GNAT (a histone acetyltransferase for H3K14), it indicated that phosphorylation of H3S10 and acetylation of H3K14 have intrinsic relationship and happen in sequence. Besides, the acetylation of H3K9 and H3K14 cooperated during expression process of salinity inducible genes. Since the PHD finger domain containing protein can also interacts with Elongin A and ISWI, H3S10Phos might also contribute much to chromatin remodeling and transcription, while H3K14Ac could regulate the gene translation by recruiting translation elongation factor Ts. / To understand the detail pattern of histone PTMs' regulation mechanism, eight peptides were employed for the PTM peptide pull-down assay using the total root nucleic proteins as input. Results demonstrated that almost all the modified peptide could recruit the rubber elongation factor protein and peroxidase 1 precursor. However, there were also noticeable interaction patterns amongst them. For example, the H3K14Ac or combined H3K14Ac with S10P (S10pK14ac) could specifically interact with histone H2B, H4 and 14-3-3 (SGF14h, belongs to non epsilon class). Besides, the soybean ascorbate peroxidase (GmAPX) was always recruited by those peptides carrying phosphorylated S10. / In order to detect the locating position of H3K14Ac on the salinity inducible genes, chromatin immunoprecipitation (ChIP) was performed using the anti-H3K14Ac antibody and the soybean chromatin total extract. H3K14Ac was found predominantly located at promoter region of GmGST gene (codes for glutathione S-transferase). Besides, the specific location of H3K14Ac on this gene, two PTMs of its adjacent cites (anti-H3K9Ac, anti-H3S10Phos) were also confirmed using ChIP assay. Our results illustrate that H3K14Ac and H3K9Ac bind to the promoter region of this gene while H3S10Phos binds to the downstream region (P4)of the GmGST gene. / Based on these findings, a scheme is proposed to illustrate the functional roles of H3 N-terminal PTMs, GmGST and GmAPX in salinity stress-induced oxidative response in soybean plant. Initially, the accumulation of cellular ROS (such as H₂O₂) could up-regulate modification of H3S10Phos and H3K14Ac, which may active the expression of GmGST. Then the increased GmGST could either directly or indirectly activate downstream targets such as GmMDAR and GmAPX through the glutathione ascorbate cycle. Since the GmAPX has antioxidant effect, the accumulation of H₂O₂ could be finally reduced. On the other hand, the over-accumulated GmAPX could transport into the nucleus and feedback to the GmGST expression system. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Pi, Erxu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Thesis committee --- p.i / Declaration --- p.ii / Abstract (in English) --- p.iii / Abstract (in Chinese) --- p.v / Acknowledgements --- p.vii / Table of contents --- p.viii / General abbreviations --- p.xii / Abbreviations of chemicals --- p.xiv / List of Tables --- p.xv / List of Figures --- p.xvi / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Soybean nutrients contribute to human diet and health --- p.1 / Chapter 1.2 --- Soybean production of the world --- p.2 / Chapter 1.3 --- Soil salinization in China --- p.4 / Chapter 1.4 --- Plant salt response insights from proteomics --- p.5 / Chapter 1.5 --- Histone post-translational modifications (PTMs) related to salinity stress --- p.7 / Chapter 1.6 --- Inheriting mechanism of histone PTMs --- p.8 / Chapter 1.7 --- Significances of the present studies --- p.9 / Chapter 1.8 --- Objectives of these studies --- p.9 / Chapter 1.9 --- Reference --- p.10 / Chapter Chapter 2 --- Insight of soybean response to salinity stress based on proteomics --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and methods --- p.19 / Chapter 2.2.1 --- Plant materials and stress treatment --- p.19 / Chapter 2.2.2 --- Protein Extraction --- p.19 / Chapter 2.2.3 --- Protein Preparation for LC-FTICR MS --- p.19 / Chapter 2.2.4 --- Analysis of Protein using Nano-LC-MS/ MS --- p.20 / Chapter 2.2.5 --- 2-DE gel electrophoresis --- p.21 / Chapter 2.2.6 --- Gel staining and image analysis --- p.21 / Chapter 2.2.7 --- Tryptic in-gel digestion --- p.21 / Chapter 2.2.8 --- Protein identification and database analysis --- p.22 / Chapter 2.2.9 --- Functional classification of proteins --- p.22 / Chapter 2.3 --- Results and discussion --- p.22 / Chapter 2.3.1 --- Construction of proteomic database --- p.22 / Chapter 2.3.2 --- Label-free quantitative analysis --- p.24 / Chapter 2.3.3 --- Discovering defense-related proteins in soybean roots --- p.25 / Chapter 2.3.4 --- Up-regulated proteins related to sense and eliminate ROS --- p.27 / Chapter 2.4 --- Reference --- p.39 / Chapter Chapter 3 --- GmPHD5 acts as an important regulator for crosstalk between histone H3K4 di-methylation and H3K14 acetylation in response to salinity stress in soybean --- p.44 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and methods --- p.45 / Chapter 3.2.1 --- Molecular cloning --- p.45 / Chapter 3.2.2 --- Expression of recombinant proteins --- p.48 / Chapter 3.2.3 --- Peptide synthesis and antibody production --- p.48 / Chapter 3.2.4 --- Nucleic protein extraction --- p.49 / Chapter 3.2.5 --- Histone protein extraction --- p.49 / Chapter 3.2.6 --- Interacting between GmPHD5 and other nuclear proteins --- p.50 / Chapter 3.2.7 --- Co-immunoprecipitation (Co-IP) and peptide pull down assays --- p.50 / Chapter 3.2.8 --- Chromatin immuno-precipitation (ChIP) assays --- p.51 / Chapter 3.2.9 --- In vitro acetyltransferase activity assay --- p.52 / Chapter 3.3 --- Results --- p.52 / Chapter 3.3.1 --- Characterization of GmPHD5 --- p.52 / Chapter 3.3.2 --- Expression of GmPHD5 in soybean --- p.54 / Chapter 3.3.3 --- GmPHD5 interacts with histone methylated H3K4 --- p.55 / Chapter 3.3.4 --- Identification of non-histone proteins that interacted with GmPHD5 --- p.57 / Chapter 3.3.5 --- Characterization of GmGNAT1 --- p.60 / Chapter 3.3.6 --- GmPHD5 interacts with GmISWI --- p.62 / Chapter 3.3.7 --- GmPHD5 located on the promoter and coding region of some salinity stress inducible genes --- p.63 / Chapter 3.4 --- Discussions --- p.64 / Chapter 3.5 --- Conclusions --- p.67 / Chapter 3.6 --- Reference --- p.68 / Chapter Chapter 4 --- Proteomics investigation of histone H3 N-terminal postranslational modifications as key regulators in response to salinity stress induced oxidative stress --- p.73 / Chapter 4.1 --- Introduction --- p.73 / Chapter 4.2 --- Materials and methods --- p.74 / Chapter 4.2.1 --- Plant materials and growth conditions --- p.74 / Chapter 4.2.2 --- Nuclei extraction and histone isolation --- p.74 / Chapter 4.2.3 --- Western blotting --- p.75 / Chapter 4.2.4 --- Chromatin immuno-precipitation (ChIP) assays --- p.75 / Chapter 4.2.5 --- Protein extraction and 2-DE --- p.76 / Chapter 4.2.6 --- Image analysis and data analysis --- p.77 / Chapter 4.2.7 --- Protein identification by MALDI-TOF/ TOF MS and database search --- p.77 / Chapter 4.2.8 --- Capillary Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance Mass Spectrometer --- p.77 / Chapter 4.3 --- Results --- p.79 / Chapter 4.3.1 --- Dynamics of genome wide histone PTMs --- p.79 / Chapter 4.3.2 --- H3K14Ac and H3K9Ac located on the promoter region of salinity stress inducible gene --- p.79 / Chapter 4.3.3 --- Nucleic proteins interacted with H3K14Ac, H3K9Ac and H3S10Phos --- p.81 / Chapter 4.3.4 --- Nucleic factors interacted with special patterns of combinatorial H3K14Ac, H3K9Ac and H3S10Phos --- p.81 / Chapter 4.3.5 --- Enzymes involved in the glutathione-ascorbate cycle increased under salinity stress --- p.87 / Chapter 4.4 --- Discussions --- p.90 / Chapter 4.5 --- Reference --- p.93 / Chapter Chapter 5 --- Conclusions and perspectives --- p.100 / Chapter 5.1 --- References --- p.106 / Supplementary files --- p.113
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328275 |
| Date | January 2012 |
| Contributors | Pi, Erxu., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource ([15], 213 leaves) : ill. (some col.), col. map |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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