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

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栽培大豆和野生大豆在很多表型上存在比較大的差異，這些差異包括種子大小，豆莢的個數，含油量和蛋白含量以及光合作用能力等。在本研究中，我們利用之前發表的31 株大豆重測序鑒定出的在栽培或者野生群體中固定的單核苷酸多態性位元點（SNP）資料進行分析，目的在於找出潛在的受選擇區域以及鑒定出受到固定SNP 位點影響的與我們關心的性狀相關的基因。我們的研究結果表明，人工選擇的強度要比自然選擇的強度大很多，同時我們利用這些SNP資料鑒定出了幾個可能受選擇的區域。我們結合已經發表的一些數量性狀（豆莢數目，種子酪氨酸含量）的QTL 的資訊，找到了一些在這些QTL 裡面可能起到主要作用的主效基因。結合KEGG 通路，我們發現了一些在植物激素信號轉导通路，植物體與病原相互作用通路，週期節律性通路，澱粉和蔗糖合成相關的通路，碳固定通路上面栽培和野生大豆差異比較大的基因，這些基因可能是人工選擇和自然選擇的產物。我們從中可以看到人工選擇和自然選擇的印記。根據在野生大豆裡面固定而在家養大豆裡面是中頻的那些SNP 的分佈情況，我們推測第四號染色體在自然選中可能具有比較大的作用。另外我們還發現第八號染色體在家養和野生之間的分化程度比較大。在所有的受SNP 位點影響的基因中，有三分之一的基因都在這條染色體上。我們進一步分析這些基因發現，很多重要農藝性狀比如矮化，根的生長，抗除草劑，抗病，抗旱等相關的基因都在這裡面。我們的這些發現對於將來高產大豆的遺傳和分子育種會有比較大的幫助。 / Cultivated and wild soybeans exhibit morphological differences in many traits, such as seed size, pod number, protein and oil content, photosynthesis capacity, etc. Here, we analyzed the fixed SNPs in both cultivated and wild soybeans reported previously by our lab for two aims: one is to search the candidate regions under selection; the other is to identify traits-related genes possessing fixed SNPs leading to non-synonymous changes. Our results indicated that artificial selection is much stronger than natural selection. We identified candidate genomic regions and genes under artificial selection, which play a vital role in several traits of interests including pod number and seed tyrosine content. By searching the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, we further discovered those important genes impacted by domestication and natural selection were enriched in several pathways including plant hormone transduction, plant-pathogen interaction, circadian rhythm, starch and sucrose metabolism, and carbon fixation pathway. Our analysis also suggested that Chromosome04 (Chr04) is important for fitness of wild accessions because that most of the residual SNPs fixed in wild soybeans showed intermediate frequency in cultivated accessions. Significantly, we observed considerable divergence between wild and cultivated accessions on Chr08. Detailed analysis indicated that nearly 30% of the genes located on Chr08 with SNPs fixed either in cultivated or wild soybeans. These genes were associated with many important agronomic traits including dwarfing, root growth, herbicide resistance, pathogen resistance, drought/salt tolerance. Our modeling results would be beneficial for genetic modification and molecule breeding in soybean in the near future. / Detailed summary in vernacular field only. / Pan, Shengkai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 85-101). / Abstracts also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.Iii / Acknowledgements --- p.iv / Table of Contents --- p.v / List of Tables --- p.viii / List of Fignres --- p.ix / Chapter CHAPTER 1 --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Morphology differences --- p.2 / Chapter 1.2 --- Herbicide --- p.4 / Chapter 1.2.1 --- Sulfonylureas herbicides --- p.4 / Chapter 1.2.2 --- Herbicide resistance --- p.5 / Chapter CHSPTER 2 --- METHODS --- p.8 / Chapter 2.1 --- SNP detection --- p.9 / Chapter Fignre 2.1. --- SOAPsnp flow chart --- p.10 / Chapter 2.2 --- Fixed SNPs detection --- p.10 / Chapter 2.2.1 --- Fixed SNPs in the wild population at low frequency (FWLC) or at intermediate frequency (FWIC) in the cultivated population --- p.11 / Chapter 2.2.2 --- Fixed SNPs in the cultivated population at low frequency (FCLW) or at intermediate frequency (FCIW) in the wild population --- p.11 / Chapter 2.3 --- InDel detection --- p.12 / Chapter 2.3.1 --- Statistics of conserved InDels in the wild population at low frequency (CWLC), intermediate (CWIC), or high frequency (CWHC) in the cultivated population --- p.13 / Chapter 2.3.2 --- Identification of conserved InDels in the cultivated population at low frequency (CCLW), intermediate (CCIW), or high frequency (CCHW) in the wild population --- p.13 / Chapter 2.4 --- Result visualization --- p.14 / Chapter CHAPTER 3 --- RESULTS --- p.15 / Chapter 3.1 --- Number of fixed SNPs and conserved InDels in cultivated accessions is much more than in wild accessions --- p.16 / Chapter 3.2 --- Candidate regions under selection --- p.24 / Chapter 3.2.1 --- Analysis of wild-unique distribution on Chromosome 04 --- p.24 / Chapter 3.2.2 --- Analysis of distribution on Chromosome 08 --- p.25 / Chapter 3.3 --- Other analysis on fixed SNPs --- p.35 / Chapter 3.3.1 --- Genes with amino acid code changes caused by SNPs fixed in the cultivated population at low frequency in wild population --- p.35 / Chapter 3.3.2 --- Genes with amino acid code changes caused by SNPs fixed in the wild population at low frequency in the cultivated population --- p.44 / Chapter 3.3.3 --- Genes with non-synonymous caused by SNPs fixed in both cultivated and wild populations --- p.56 / Chapter 3.3.4 --- Analysis combining KEGG pathway with genes containing amino acid code change sites resulted from fixed SNPs --- p.60 / Chapter CHAPTER 4 --- DISCUSSION --- p.75 / Chapter 4.1 --- Differences between cultivated and wild soybeans --- p.76 / Chapter 4.2 --- Roles of fixed SNPs in the wild and cultivated populations study --- p.76 / Chapter 4.3 --- The possible reason for number of fixed SNPs and conserved InDels in cultivated soybeans is much more than that in wild soybeans --- p.77 / Chapter 4.3.1 --- Artificial selection is much stronger than natural selection --- p.77 / Chapter 4.3.2 --- Bottleneck in domestication process of cultivated soy bean --- p.78 / Chapter 4.4 --- Notes about fixed SNPs in the gene regions --- p.79 / Chapter 4.5 --- Possible reason for divergence in Chromosome 08 --- p.79 / Chapter 4.6 --- Possible reason for unique pattern of SNPs fixed in wild accessions on Chromosome 04 --- p.80 / Chapter CHAPTER 5 --- PROSPECTIVE --- p.81 / REFERENCES --- p.84

In order to identify plant genes involved in the Rhizobium/legume root nodule symbiosis, host gene expression during soybean nodule development was studied. The hybridization of complementary DNA (cDNA) probes with homologous and heterologous polyadenylated polysomal RNAs showed that most of the 20,000-25,000 RNA sequences expressed were common to uninfected root and nodule tissues. There was a marked increase in the relative concentration of leghaemoglobin and moderately abundant nodule sequences following infection by Rhizobium. A nodule-specific cDNA probe (NS-cDNA), prepared by eliminating common root sequences by cascade hydroxylapatite chromatography, was used to characterize a small population of nodule-specific sequences. Hybridization of NS-cDNA to DNA from soybean embryos and not to Rhizobium demonstrated that these sequences are encoded by the host genome. Hybridization with nuclear RNA from uninfected tissues showed that the primary mode of regulation of these sequences is at the level of transcription. Expression of these small nodule-specific-mRNAs is differentially modulated by ineffective strains of Rhizobium. The relative concentration of nodule-specific and common moderately abundant sequences increased to varying extents, in parallel with leghaemoglobin, reaching a peak when nitrogen fixation commences. These data suggest that host gene expression is coordinately regulated during early nodule development. Indoleacetic acid appeared to modulate the expression of common moderately abundance sequences, but had no detectable effect upon leghaemoglobin or nodule-specific sequences. Preliminary characterization of 26 cDNA clones indicated that, in addition to leghaemoglobin and other positively regulated sequences, there were two clones whose mRNA concentration decreased during nodule development.

No description available.

鹽鹼化是世界上最主要非生物脅迫之一，它主要是由於土壤中的鹽（氯化鈉）過量積累所導致的, 不僅影響植物的生長而且也影響農作物的產量。大豆是世界上最重要的經濟類豆科植物之一，由於其種子內含有豐富的營養物質例如蛋白質，油，糖分和纖維，所以它為我們提供了極為重要和大量的油脂和蛋白。在鹽脅迫下大豆的產量會有明顯的降低。由於以上這些情況，我們希望搞清楚大豆對於鹽脅迫的反應機制。首先是通過蛋白質組學弄清楚鹽脅迫的生理過程和大豆如何耐受鹽脅迫的。一般來說，蛋白質組學包括了鑑定蛋白類的化合物和測量在生物系統中的蛋白含量的學科。近期，質譜的發展提供了一個去研究細胞內蛋白質的動態變化十分有用的平台。定量蛋白質組學的發展對於我們系統性的了解蛋白質的功能的分子是十分重要的並且預期會提供給我們各種生物過程和系統的分子機制的見解.其次，表觀遺傳性特別是組蛋白修飾。因為組蛋白修飾通過重新編排染色體的結構和組成參與了許多重要的生物過程並且這些翻譯後修飾對於植物的耐受鹽脅迫也發揮著十分重要的作用。因此我們希望了解組蛋白修飾是如何參與這一過程的。 / Salinity stress, which is caused by the accumulation of excessive amount of salts in the soil, is one of the most severe abiotic stresses that constraint not only crop plant growth but also crop productivity. Soybean (Glycine max) is one of the most important economical legume crops in the world because of its richness of nutritional compositions including protein, oil, sugar and fiber in the seed. Soybean yield of sensitive cultivars is decreased dramatically under salt stress. Because of this, we tried to figure out the mechanism of how soybean response to salinity stress. Firstly, Proteomics--to elucidate the affected physiological process in the salinity stress and the way to tolerant the stress. In general, proteomics involves the identification of protein components and the measurement of protein abundance in biological systems. Recent mass spectrometry (MS)-based technology developments have provided useful platforms for the study of quantitative changes in protein components within the cell. Quantitative proteomics is important for the system-based understanding of the molecular function of each protein component and expected to provide insights into molecular mechanisms of various biological processes and systems. Secondly, Epigenetics--particularly histone modifications. Because histone modifications play important roles in many fundamental biological processes by rearranging the structure and composition of chromatin and PTMs have more roles in response salinity stress. So we want to understand how PTMs involve in this process from epigenetics. / Detailed summary in vernacular field only. / Peng, Xu. / "October 2012." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 44-54). / Abstracts also in Chinese. / Thesis committee --- p.i / Declaration --- p.ii / Abstract (in English) --- p.iii / Abstract (in Chinese) --- p.iv / Acknowledgements --- p.v / Table of contents --- p.vi / General abbreviations --- p.vii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Soybean --- p.1 / Chapter 1.2 --- Salinity stress and plants’ response to salinity stress --- p.1 / Chapter 1.3 --- Mass Spectrometry base Proteomics --- p.3 / Chapter 1.3.1 --- Introduction to proteomics --- p.3 / Chapter 1.3.2 --- Proteomic studies in plants --- p.5 / Chapter 1.4 --- Introduction to Epigenetics --- p.6 / Chapter 1.5 --- Present studies --- p.8 / Chapter Chapter 2 --- Proteomic studies in soybean --- p.9 / Chapter 2.1 --- Materials and methods --- p.9 / Chapter 2.1.1 --- Plant materials and stress treatment --- p.9 / Chapter 2.1.2 --- Protein extraction and nuclei extraction and histone isolation --- p.10 / Chapter 2.1.3 --- Protein Preparation for Mass Spectrometry --- p.11 / Chapter 2.1.4 --- Analysis of Protein using nanoLC-MS/MS and Data analysis --- p.12 / Chapter 2.2 --- Results and discussions --- p.13 / Chapter 2.2.1 --- One thousand two hundred seventeen proteins were identified by LC MS/MS-based proteomics technique --- p.13 / Chapter 2.2.2 --- Functional analyses of identified proteins --- p.22 / Chapter 2.2.3 --- One hundred sixty-three proteins are changed under salinity stress as identified by LC MS/MS-based proteomics technique --- p.26 / Chapter 2.2.4 --- Important stress relate proteins were identified by LC MS/MS-based proteomics technique --- p.30 / Chapter 2.2.5 --- Histone PTMs in soybean under salinity stress --- p.34 / Chapter Chapter 3 --- Conclusions and perspectives --- p.35

BURP域家族是植物特有的一個蛋白家族，結構多樣，共同點是在羧基端都有一個保守的BURP域。迄今為止，關於BURP域家族成員的功能及細胞定位的研究非常有限。部分RD22-like的亞家族成員由於顯示出受非生物脅迫而誘導表達的特性，因此被認為功能可能與非生物脅迫回應相關。本研究對克隆到的一個受非生物脅迫誘導表達的大豆基因(GmRD22)進行了生物進化分析，并詳細分析了其在不同的大豆品種以及不同非生物脅迫下的表達模式，揭示了其表達豐度與大豆的抗非生物脅迫能力有關，並且使用不同的轉基因系統(細胞水準跟植物水準)揭示了其過量表達有助於減輕非生物脅迫對植物造成的影響。研究利用GFP融合蛋白追蹤技術和免疫電鏡技術揭示GmRD22蛋白定位於細胞壁，其中BURP域對於GmRD22定位于細胞壁起到關鍵作用。研究也揭示了GmRD22能夠與細胞外的一種過氧化物酶GmPer1相互作用，GmRD22在轉基因擬南芥和轉基因水稻中的過量表達能夠顯著提高脅迫條件下轉基因植株中木質素的含量。我們認為GmRD22通過與細胞壁過氧化物酶的相互作用來提高植物在脅迫條件下細胞壁的完整性從而增強植株的抗性。 / The BURP-domain protein family comprises a diverse group of plant-specific proteins that share a conserved BURP domain at the C terminus. However, there have been only limited studies on the functions and subcellular localization of these proteins. Members of the RD22-like subfamily are postulated to associate with stress responses due to the stress-inducible nature of some RD22-like genes. In this report, different expression patterns of a stress-inducible RD22-like protein from soybean (GmRD22) either in different soybean species or under different osmotic stress conditions were analyzed, different transgenic systems (cells and in planta) were used to show that the ectopic expression of GmRD22 can alleviate salinity and osmotic stress. The detailed microscopic studies were also performed using both fusion proteins and immuno-electron microscopic techniques to demonstrate the apoplast localization of GmRD22, for which the BURP domain is a critical determinant of the subcellular localization. The apoplastic GmRD22 interacts with a cell wall peroxidase and the ectopic expression of GmRD22 in both transgenic A. thaliana and transgenic rice resulted in increased lignin production when subjected to salinity stress. It is possible that GmRD22 regulates cell wall peroxidase and hence strengthens cell wall integrity under osmotic stress conditions. / Detailed summary in vernacular field only. / Wang, Hongmei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 124-136). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iii / Table of contents --- p.v / List of tables --- p.x / List of figures --- p.xi / General abbreviations --- p.xiii / Chemical abbreviations --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Abiotic stress in the world --- p.2 / Chapter 1.2 --- The advances of plant abiotic stress resistance mechanisms --- p.5 / Chapter 1.2.1 --- Sensor of salt stress --- p.7 / Chapter 1.2.2 --- Reestablishment of ionic homeostasis --- p.9 / Chapter 1.2.3 --- Osmoregulation by compatible osmolytes --- p.11 / Chapter 1.2.4 --- Oxidative stress management --- p.12 / Chapter 1.2.5 --- Transcription regulation of gene expression in osmotic stress --- p.14 / Chapter 1.3 --- The BURP-domain protein family --- p.19 / Chapter 1.3.1 --- Introduction of BURP-domain protein family --- p.19 / Chapter 1.3.2 --- Advances of BURP-domain protein studies --- p.20 / Chapter 1.3.3 --- The BURP-domain protein and osmotic stress --- p.21 / Chapter 1.4 --- Background information of this project --- p.22 / Chapter 1.5 --- Hypothesis and objectives --- p.25 / Chapter Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Bacterial strains, vectors and plasmids, cell lines and plant materials --- p.27 / Chapter 2.2 --- Chemicals and reagents --- p.32 / Chapter 2.3 --- Primers used in this study --- p.35 / Chapter 2.4 --- Molecular cloning of GmRD22 --- p.38 / Chapter 2.5 --- DNA and RNA extraction and Northern blot --- p.40 / Chapter 2.5.1 --- DNA and plasmid extraction --- p.40 / Chapter 2.5.2 --- RNA extraction from plant --- p.41 / Chapter 2.5.3 --- Generation of DIG-labeled PCR probe --- p.41 / Chapter 2.5.4 --- Northern blot --- p.43 / Chapter 2.6 --- Reverse transcription and Real-time analysis --- p.44 / Chapter 2.7 --- Phylogenetic analysis --- p.45 / Chapter 2.8 --- Basic molecular techniques --- p.46 / Chapter 2.8.1 --- Recombinant DNA --- p.46 / Chapter 2.8.2 --- Transformation of E. coli competent cells --- p.46 / Chapter 2.8.3 --- Transformation of A. tumefacien competent cells --- p.47 / Chapter 2.8.4 --- Gel electrophoresis --- p.48 / Chapter 2.8.5 --- Sequencing --- p.48 / Chapter 2.9 --- Establishment of transgenic models --- p.49 / Chapter 2.9.1 --- Establishment of transgenic BY-2 cell --- p.49 / Chapter 2.9.2 --- Establishment of transgenic A. thaliana --- p.50 / Chapter 2.9.3 --- Establishment of transgenic rice --- p.51 / Chapter 2.10 --- Cell viability assay under osmotic stress treatment --- p.51 / Chapter 2.11 --- Root elongation assay of transgenic A. thaliana --- p.52 / Chapter 2.12 --- Osmotic stresses treatment of transgenic rice lines --- p.52 / Chapter 2.13 --- Protein expression, production of antibodies and Western blot --- p.53 / Chapter 2.14 --- Subcellular localization of fusion protein by confocal microscopic study --- p.55 / Chapter 2.15 --- Electron microscopic study --- p.56 / Chapter 2.16 --- Immunoprecipitation and mass spectrometry --- p.57 / Chapter 2.17 --- Cell wall components analysis --- p.60 / Chapter 2.18 --- Statistical analysis --- p.61 / Chapter Chapter 3 --- Results --- p.62 / Chapter 3.1 --- GmRD22 gene --- p.63 / Chapter 3.1.1 --- GmRD22 encodes a BURP-domain protein in soybean --- p.63 / Chapter 3.1.2 --- Phylogenetic analysis of GmRD22 --- p.65 / Chapter 3.2 --- GmRD22 gene expression --- p.73 / Chapter 3.2.1 --- GmRD22 shows a biphasic induction by salinity stress and ABA treatment --- p.73 / Chapter 3.2.2 --- GmRD22 is also inducible by osmotic stress --- p.76 / Chapter 3.2.3 --- GmRD22 shows stronger and faster induction in WF 7 than Union --- p.76 / Chapter 3.3 --- Functional study --- p.78 / Chapter 3.3.1 --- Construction of GmRD22 transformants --- p.78 / Chapter 3.3.2 --- Ectopic expression of GmRD22 improve osmotic stresses tolerance in transgenic BY-2 cells --- p.80 / Chapter 3.3.3 --- Ectopic expression of GmRD22 alleviates osmotic stresses in transgenic A. thaliana --- p.83 / Chapter 3.3.4 --- Ectopic expression of GmRD22 alleviates osmotic stresses in transgenic rice --- p.86 / Chapter 3.4 --- GmRD22 is an apoplastic protein --- p.90 / Chapter 3.4.1 --- Western blot analysis in different soybean extracts --- p.90 / Chapter 3.4.2 --- Subcellular localization of GmRD22-GFP fusion protein in onion epidermal and A. thaliana root system --- p.93 / Chapter 3.4.3 --- GmRD22 localization in native soybean --- p.96 / Chapter 3.5 --- BURP domain is essential for the subcellular localization --- p.99 / Chapter 3.6 --- GmRD22 interacts with a putative apoplastic peroxidase --- p.103 / Chapter 3.6.1 --- Identification of GmRD22 interacting protein --- p.103 / Chapter 3.6.2 --- GmPer1 is a putative extracellular class III peroxidase --- p.107 / Chapter 3.6.3 --- Overexpression of GmRD22 affected lignin metabolism in transgenic rice and A. thaliana under salinity stress --- p.109 / Chapter 3.6.4 --- GmPer1 homologues increased expression under salinity stress --- p.113 / Chapter Chapter 4 --- Discussion and Conclusion --- p.115 / Chapter 4.1 --- GmRD22 as a member of RD22-like subfamily --- p.116 / Chapter 4.2 --- Induction mechanism of GmRD22 expression is related to ABA --- p.117 / Chapter 4.3 --- Biological function of GmRD22 providing protective effect under osmotic stress --- p.118 / Chapter 4.4 --- The BURP domain of GmRD22 plays a key role in its apoplastic targeting --- p.119 / Chapter 4.5 --- The interaction between GmRD22 and apoplastic peroxidase provides the clue for the mechanism of enhanced osmotic stress tolerance in GmRD22 transgenic plants --- p.120 / Chapter 4.6 --- Conclusion --- p.123 / References --- p.124 / Chapter Appendix I --- Restriction and modifying enzymes --- p.137 / Chapter Appendix II --- Chemicals --- p.138 / Chapter Appendix III --- Buffer, solution, gel and medium formulation --- p.143 / Chapter Appendix IV --- Equipment and facilities --- p.146

大豆的豐富營養和經濟價值使它成為重要的農產品。但是，土壤鹽漬化影響著大豆的產量。這個問題在沿岸地方特別嚴重。若要改善大豆的耐鹽能力，必先增加對大豆耐鹽機理的了解。 / 本實驗室從大豆中發現了一個受鹽脅迫誘導表達的基因GmSAL1。以往透過體外酶反應分析法， GmSAL1蛋白被介定為一個能作用於1,4,5-三磷酸肌醇 (IP₃) 的肌醇磷酸-5-磷酸酶。這有別於在擬南芥中的SAL1同源蛋白AtSAL1, AtSAL1是一個肌醇磷酸-1-磷酸酶。由於IP₃是信號傳導途徑中的重要分子，本課題對與IP₃信號及耐鹽性相關的GmSAL1蛋白功能進行研究。 / 本課題旨在：（一）　研究GmSAL1對於細胞質內IP₃的累積的體內作用； （二） 研究 GmSAL1 在脫落酸 (ABA)　的信號傳導中的可能角色； （三） 研究 GmSAL1 在鹽脅迫下的保護作用。 / 本研究利用體內報告系統證明了 GmSAL1 對於減少細胞質內IP₃的累積的作用。這種影響IP₃水平的功能減弱了由 ABA 信號所引起的氣孔關閉和種子萌發抑制。利用 GmSAL1 轉基因煙草細胞 (BY-2)　和擬南芥，證明 GmSAL1 在鹽脅迫下起著短暫的保護作用。GmSAL1在鹽脅迫下的保護功能可能是由於蒸騰作用的局部恢復和細胞鈉離子區室化的作用。 / 本研究展示了肌醇信號，ABA信號和鹽脅迫反應三者之間的關係。這是在以前的研究中未被清楚闡釋的。 / Soybean is nutritionally and economically important. However, high soil salinity, particularly in coastal regions, impedes the production of soybean. Understanding the salt tolerance mechanism is the first step towards the enhancement of salt tolerance of soybean. / Our laboratory identified a salt-responsive gene from soybean namely GmSAL1. Previous in vitro enzyme assay suggested that the GmSAL1 protein is an inositol 5’-phopsphatase acting on inositol 1,4,5-trisphosphate (IP₃), which is different from the enzymatic activity reported for the SAL1 homologue (AtSAL1) in Arabidopsis thaliana (A. thaliana) which is an inositol 1-phopsphatase. Since IP₃ is an important molecule involved in signal transduction pathways, this project is to explore the in vivo functions of GmSAL1 in relation to IP3 signaling and salinity tolerance. / The specific objectives of this research are (1) to study the in vivo role of GmSAL1 on cytosolic IP₃ accumulation; (2) to study the possible involvement of GmSAL1 in ABA signaling; (3) to study the protective roles of GmSAL1 under salt stress. / In this project, the function of GmSAL1 to reduce cytosolic IP₃ was demonstrated using an in vivo reporter system. This activity on IP₃ levels reduced the sensitivity of stomatal closure and seed germination inhibition mediated by ABA signals. A transient protection effect against the ionic effect under salt stress by GmSAL1 was shown by gain-of-function tests in transgenic BY-2 cells and transgenic A. thaliana. The protective effect conferred by GmSAL1 may be due to a partial resuming of transpiration through reduction of ABA signals and compartmentalization of Na⁺ into vacuoles. / The study of GmSAL1 in this research demonstrated the link among inositol signaling, ABA signaling, and salinity response which was not well addressed in previous reports. / 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. / Ku, Yee Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 97-104). / Abstracts also in Chinese. / Statement --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgements --- p.v / General Abbreviations --- p.vii / Abbreviations of Chemicals --- p.ix / Table of Contents --- p.xi / List of Figures --- p.xviii / List of Tables --- p.xxi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction to salinity and agriculture --- p.1 / Chapter 1.1.1 --- Adverse effects of salinity on plants --- p.2 / Chapter 1.1.1.1 --- Osmotic stress --- p.2 / Chapter 1.1.1.2 --- Ionic stress --- p.3 / Chapter 1.1.1.3 --- Separation of ionic effect from osmotic effect --- p.3 / Chapter 1.1.1.4 --- Oxidative stress --- p.4 / Chapter 1.1.2 --- Major physiological responses of plants to achieve salt tolerance --- p.5 / Chapter 1.1.2.1 --- Maintenance of cellular ion homeostasis --- p.5 / Chapter 1.1.2.2 --- Balance between Na⁺ and K⁺ influx --- p.5 / Chapter 1.1.2.3 --- Efflux of Na⁺ from cell --- p.9 / Chapter 1.1.2.4 --- Enhanced compartmentalization of Na⁺ and Cl⁻ in vacuole --- p.11 / Chapter 1.1.2.5 --- Enhanced vacuolar ion compartmentalization --- p.13 / Chapter 1.1.2.6 --- Biosynthesis of osmolytes --- p.13 / Chapter 1.2 --- Signal transduction under salt stress --- p.14 / Chapter 1.2.1 --- General introduction to signal transduction under salt stress --- p.14 / Chapter 1.2.2 --- ABA signaling under salinity --- p.15 / Chapter 1.2.2.1 --- General introduction to ABA signaling --- p.15 / Chapter 1.2.2.2 --- IP₃ and ABA signaling --- p.16 / Chapter 1.2.2.3 --- Introduction to inositol phosphate --- p.16 / Chapter 1.2.2.4 --- Phosphatidylinositol-3-monophosphate --- p.18 / Chapter 1.2.2.5 --- Phosphatidylinositol-4-monophosphate --- p.18 / Chapter 1.2.2.6 --- Phosphatidylinositol-5-monophosphate --- p.18 / Chapter 1.2.2.7 --- Phosphatidylinositol (3,5) bisphosphate --- p.19 / Chapter 1.2.2.8 --- Phosphatidylinositol (4,5) bisphosphate --- p.19 / Chapter 1.2.2.9 --- Inositol (1,4,5) trisphosphate (IP₃) --- p.19 / Chapter 1.2.2.10 --- Inositol metabolism under salt stress --- p.19 / Chapter 1.2.2.11 --- The involvement of IP₃ in ABA signaling --- p.20 / Chapter 1.2.3 --- General introduction to Ca²⁺ signaling --- p.22 / Chapter 1.2.4 --- Ca²⁺ channels --- p.23 / Chapter 1.2.4.1 --- Ligand-gated Ca²⁺ channels --- p.23 / Chapter 1.2.4.1.1 --- IP₃ gated Ca²⁺ channels --- p.23 / Chapter 1.2.4.1.2 --- Cyclic nucleotide gated channels (CNGCs) --- p.24 / Chapter 1.2.4.1.3 --- Glutamate receptor homologs (GLRs) --- p.24 / Chapter 1.2.4.2 --- Voltage-gated Ca2⁺ channels --- p.25 / Chapter 1.2.4.2.1 --- Two-pore channels (TPCs) --- p.25 / Chapter 1.2.4.2.2 --- Mechanosensitive Ca2²⁺permeable channels (MSCCs) --- p.25 / Chapter 1.2.4.2.3 --- Ca²⁺ and ABA signaling --- p.26 / Chapter 1.2.5 --- ABA, IP₃ and Ca²⁺ signaling --- p.26 / Chapter 1.2.5.1 --- Ca²⁺ signaling under salt stress --- p.30 / Chapter 1.2.5.2 --- Ca²⁺ signal mediated cellular responses --- p.30 / Chapter 1.3 --- Introduction to inositol phosphatases --- p.30 / Chapter 1.3.1 --- Previous studies on inositol phosphatases in plant --- p.33 / Chapter 1.3.1.1 --- Inositol polyphosphate 1-phosphatase --- p.33 / Chapter 1.3.1.2 --- Inositol polyphosphate 5-phosphatase --- p.34 / Chapter 1.4 --- Previous research on GmSAL1 in Prof. Hon-Ming Lam’s lab --- p.37 / Chapter 1.5 --- Objective and Significance of this project --- p.38 / Chapter Chapter 2 --- Materials and methods --- p.39 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Plants, bacterial strains and vectors --- p.39 / Chapter 2.1.2 --- Chemicals and enzymes --- p.40 / Chapter 2.1.3 --- Buffer, medium and solution --- p.41 / Chapter 2.1.4 --- Primers --- p.41 / Chapter 2.1.5 --- Equipments and facilities --- p.44 / Chapter 2.1.6 --- Software --- p.44 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Measurement of osmolarity --- p.44 / Chapter 2.2.2 --- Plant growth and treatment conditions --- p.45 / Chapter 2.2.2.1 --- NaCl, PEG and ABA treatment on soybean plant --- p.45 / Chapter 2.2.3 --- Artificial crossing of A. thaliana --- p.46 / Chapter 2.2.3.1 --- Screening of double homozygous transgenic A. thaliana lines --- p.46 / Chapter 2.2.4 --- Transformation of tobacco BY-2 cells --- p.47 / Chapter 2.3 --- Molecular techniques --- p.48 / Chapter 2.3.1 --- DNA extraction --- p.48 / Chapter 2.3.2 --- PCR --- p.48 / Chapter 2.3.2.1 --- Screening of transgenes --- p.48 / Chapter 2.3.2.2 --- Synthesis of DIG-labeled DNA probe for northern blot analysis --- p.49 / Chapter 2.3.3 --- DNA gel electrophoresis --- p.49 / Chapter 2.3.4 --- RNA extraction --- p.50 / Chapter 2.3.5 --- Northern blot analysis --- p.51 / Chapter 2.3.5.1 --- RNA treatment --- p.51 / Chapter 2.3.5.2 --- Electrophoresis --- p.51 / Chapter 2.3.5.3 --- RNA blotting --- p.51 / Chapter 2.3.5.4 --- GmSAL1 mRNA detection --- p.52 / Chapter 2.4 --- Cell viability assay --- p.52 / Chapter 2.5 --- Na⁺ compartmentalization assay --- p.53 / Chapter 2.6 --- ABA sensitivity assays --- p.53 / Chapter 2.6.1 --- Seed germination assay --- p.53 / Chapter 2.6.2 --- Stomatal opening assay --- p.54 / Chapter Chapter 3 --- Results --- p.55 / Chapter 3.1 --- Differential response of GmSAL1 expression level to NaCl and PEG treatment --- p.55 / Chapter 3.2 --- The expression of GmSAL1 in host plant is responsive to ABA --- p.59 / Chapter 3.3 --- Effect of GmSAL1 on cytosolic IP₃ level in vivo --- p.62 / Chapter 3.4 --- Overexpression of GmSAL1 down-regulates in planta IP₃ level in guard cell --- p.62 / Chapter 3.5 --- Ectopic expression of GmSAL1 in A. thaliana alters stomatal aperture in the presence of ABA in a Ca²⁺ dependent manner --- p.67 / Chapter 3.6 --- Ectopic expression of GmSAL1 in A. thaliana reduces the ABA inhibitory effect on seed germination --- p.71 / Chapter 3.7 --- Overexpression of GmSAL1 transiently protects A. thaliana against ionic effect under salinity --- p.76 / Chapter 3.8 --- Overexpression of GmSAL1 enhances the survival of tobacco BY-2 cells under salt treatment but not near iso-osmotic PEG treatment --- p.80 / Chapter 3.9 --- Overexpression of GmSAL1 confers enhanced vacuolar compartmentalization of Na⁺ in NaCl treated BY-2 cells --- p.85 / Chapter Chapter 4 --- Discussion --- p.90 / Chapter 4.1 --- GmSAL1 as a novel inositol 5-phosphatase --- p.90 / Chapter 4.2 --- The effect of GmSAL1 expression on ABA signaling --- p.91 / Chapter 4.3 --- Involvement of GmSAL1 in tolerance toward ionic effect under salt stress --- p.92 / Chapter 4.4 --- The protective function of GmSAL1 under salinity --- p.93 / Chapter Chapter 5 --- Conclusion --- p.96 / References --- p.97 / Chapter Appendix I --- Chemicals --- p.105 / Chapter Appendix II --- Formulations of buffer, medium and solution --- p.107 / Chapter Appendix III --- Equipments and facilities --- p.110 / Chapter Appendix IV --- Osmolarity of solutions --- p.111 / Chapter Appendix V --- Result of biological repeat of northern blot analysis of GmSAL1 in soybean leaf under NaCl --- p.113 / Chapter Appendix VI --- Result of biological repeat of northern blot analysis of GmSAL1 in soybean root under NaCl --- p.114 / Chapter Appendix VII --- Result of biological repeat of northern blot analysis of GmSAL1 in soybean leaves under 100μM ABA treatment for 0hr, 0.5hr, 1hr, 2hr and 4hr --- p.115 / Chapter Appendix VIII --- Result of biological repeat experiment on the survival rate of tobacco BY-2 cell in 150mM NaCl supplemented MS medium --- p.116 / Chapter Appendix IX --- Result of biological repeat experiment on the survival rate of tobacco BY-2 cell in 13.3% PEG-6000 supplemented MS medium --- p.117

No description available.