Fósforo (P) é um dos principais nutrientes que limitam a produção vegetal. Em arabidopsis, sua deficiência reduz o comprimento da raiz principal e aumenta o número e a densidade de raízes laterais. O isolamento e caracterização de mutantes têm ajudado a elucidar a função de genes envolvidos na superação da deficiência de P. Este estudo objetivou fazer a caracterização de respostas morfo-fisiológicas dos mutantes de Arabidopsis thaliana p9, p23 e p37 a suprimentos contrastantes de P, bem como avaliar interações nutricionais de P com outros nutrientes. Este trabalho foi desenvolvido em colaboração com a University of California at Davis, Davis - Califórnia, EUA. Todos os estudos foram conduzidos em câmara de crescimento. Entre os estudos conduzidos citam-se: efeito de formulações de meios de cultura na arquitetura radical; caracterização morfo-fisiológica dos mutantes; transferência de plantas entre meios de cultura com contrastes de P e nitrogênio (N); respostas as interações nutricionais P x Fe e P x N. A maioria das avaliações centraram-se no desenvolvimento do sistema radical, porém em alguns estudos, analisou-se a expressão de genes de resposta à limitação por P. A presença de ácidos nucléicos no meio de cultura reduz o desenvolvimento radical dos três mutantes, sobretudo em p9. A ausência de Fe no meio permite resgate do fenótipo radical de COL em p9, enquanto a supressão de N possibilita resgate do fenótipo em p23 e p37, independente da condição de P. A inibição radical em arabidopsis causada pelo Fe é agravada sob deficiência de P. Parte do fenótipo radical dos mutantes pode ser causada por defeitos na síntese e/ou sinalização de auxinas ou citocininas. As mutações de p9 e p23 foram localizadas no braço superior do cromossomo 1, próximas ao marcador F23M19 onde se obteve as menores taxas de recombinação (0,0% - p9Ler e 7,8% - p23Ler). / Phosphorus (P) is one of the main limiting nutrients to plant production. In arabidopsis, P deficiency reduces the primary root length and increases the number and the density of lateral roots. Isolation and characterization of mutants have contributed to better understanding the function of several genes involved in overcoming P starvation. This study has had as objective figure out morpho-physiological response of the p9, p23 and p37 Arabidopsis thaliana mutants in different P supply conditions, as well as evaluates and identify nutritional interactions between P and other nutrients. This work was developed in a collaborative work with the University of California at Davis, Davis - California, U.S.A. All studies were carried out in growth chamber. Among the conducted studies there are: effect of media on the root arquitecture; morphological and physiological characterization of the mutants; studies with plant transference from media with different P and nitrogen (N) levels and check for nutritional interactions between P x Fe and P x N. Most of the evaluations were focused on root development. However, in some studies we also analyzed the expression of genes related to P limitation. The presence of nucleic acids in the growth media reduces root development of the three mutants, particularly in p9. The absence of Fe in the media rescues the COL root phenotype in p9, while N suppression rescues that phenotype in p23 and p37, regardless of the P condition. The root inhibition in arabidopsis caused by Fe is stronger under P deficiency. Part of the mutant root phenotype might be caused by defects in the synthesis and/or signaling of auxins or cytokinins. The p9 and p23 mutations were mapped to the upper arm of chromosome 1, next to marker F23M19 for which the lowest recombination ratios were obtained (0.0% - p9Ler and 7.8% - p23Ler).

Wong, Hon-Kit. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 119-139). / Abstract in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vii / General abbreviations --- p.ix / Abbreviations of chemicals --- p.x / List of figures --- p.xii / Table of contents --- p.xvi / Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Nitrogen assimilation and regulation in plants --- p.1 / Chapter 1.2 --- Asparagine metabolism and its gene regulation in plants --- p.2 / Chapter 1.2.1 --- A brief introduction of asparagine --- p.2 / Chapter 1.2.2 --- Asparagine synthetase gene family in A. thaliana --- p.3 / Chapter 1.2.3 --- Reciprocal regulation of ASN1 and ASN2 gene --- p.3 / Chapter 1.2.4 --- Primary structure difference of ASN1 and ASN2 protein --- p.4 / Chapter 1.2.5 --- "ASN1 overexpressor support the notion that it is a major gene regulating the free asparagine levels in plant tissues, while ASN may play different physiological function(s)" --- p.2 / Chapter 1.2.6 --- Evidence support ammonium-dependent AS in plant --- p.6 / Chapter 1.3 --- Ammonium toxicity and mechanism of ammonium toxicity to plant --- p.7 / Chapter 1.3.1 --- Ammonium toxicity --- p.7 / Chapter 1.3.2 --- Mechanism of ammonium toxicity --- p.9 / Chapter 1.4 --- "Relationship among asparagine, ammonium, and stress physiology" --- p.12 / Chapter 1.4.1 --- Ammonium accumulates under stress conditions --- p.12 / Chapter 1.4.2 --- Asparagine accumulates under stress conditions --- p.14 / Chapter 1.5 --- Relationship of asparagine metabolism and photorespiration --- p.17 / Chapter 1.5.1 --- A brief introduction of photorespiratory pathway --- p.17 / Chapter 1.5.2 --- Involvement of Asn in the photorespiration nitrogen cycle --- p.18 / Chapter 1.5.3 --- Reassimilation of ammonium released from photorespiration --- p.19 / Chapter 1.5.4 --- Photorespiration and stress physiology --- p.21 / Chapter 1.6 --- Role of amino acids in abiotic stress resistance --- p.23 / Chapter 1.6.1 --- Overview --- p.23 / Chapter 1.6.2 --- Proline accumulation and plant adaptation to water deficits and salinity stress --- p.24 / Chapter 1.6.3 --- Role of amino acids as precursors of quaternary ammonium compounds serving as compatible osmolytes --- p.28 / Chapter 1.7 --- A brief history of protoplast transient expression systems --- p.35 / Chapter 1.8 --- Advantages of mesophyll protoplast transient expression systems --- p.37 / Chapter 1.9 --- Hypothesis and main idea of this study --- p.38 / Chapter 2 --- Methods and Materials --- p.39 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Plants --- p.39 / Chapter 2.1.2 --- Bacterial strains and plasmid vector --- p.39 / Chapter 2.1.3 --- Primer used --- p.39 / Chapter 2.1.4 --- Chemicals and reagents used --- p.40 / Chapter 2.1.5 --- Solution used --- p.40 / Chapter 2.1.6 --- Commercial kits used --- p.40 / Chapter 2.1.7 --- Equipment and facilities used --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Growth medium and condition --- p.41 / Chapter 2.2.1.1 --- Normal growth condition --- p.41 / Chapter 2.2.1.2 --- Growth medium and stresses treatments --- p.41 / Chapter 2.2.1.3 --- Plant growth in Azaserine medium --- p.43 / Chapter 2.2.2 --- Biochemical Assay --- p.44 / Chapter 2.2.2.1 --- Ammonium assay --- p.44 / Chapter 2.2.2.2 --- Ammonium extraction for ammonium assay --- p.46 / Chapter 2.2.2.3 --- Soluble protein determination --- p.46 / Chapter 2.2.2.4 --- Detection of chlorophyll content --- p.47 / Chapter 2.2.3 --- Molecular techniques --- p.47 / Chapter 2.2.3.1 --- Bacterial cultures for recombinant DNA --- p.47 / Chapter 2.2.3.2 --- Recombinant DNA techniques --- p.48 / Chapter 2.2.3.3 --- Transformation of DH5a Competent cell --- p.48 / Chapter 2.2.3.4 --- Gel electrophoresis --- p.49 / Chapter 2.2.3.5 --- DNA and RNA extraction from plant tissues --- p.50 / Chapter 2.2.3.6 --- Generation of cRNA probes for Northern blot analyses --- p.52 / Chapter 2.2.3.7 --- Northern blot analysis --- p.53 / Chapter 2.2.3.8 --- PCR techniques --- p.54 / Chapter 2.2.3.9 --- Sequencing --- p.55 / Chapter 2.2.4 --- Genetic techniques --- p.56 / Chapter 2.2.4.1 --- Backcross of Azaserine resistant mutant --- p.56 / Chapter 2.2.4.2 --- Phenotype screening of backcross progenies --- p.56 / Chapter 2.2.5 --- Transient gene expression --- p.57 / Chapter 2.2.5.1 --- Protoplast isolation from Arabidopsis leave --- p.57 / Chapter 2.2.5.2 --- Protoplast transformation --- p.58 / Chapter 2.2.5.3 --- Gus protein extraction from protoplasts --- p.59 / Chapter 2.2.5.4 --- Gus assay --- p.60 / Chapter 2.2.5.5 --- MU calibration standard --- p.60 / Chapter 2.2.5.6 --- Sample assay --- p.60 / Chapter 3 --- Result --- p.61 / Chapter 3.1 --- Expression of ASN2 and ammonium assay in Arabidopsis thaliana under various stress conditions and senescence --- p.61 / Chapter 3.1.1 --- Ammonium assay of wild type seedlings under stress conditions --- p.61 / Chapter 3.1.2 --- Kinetic studies of ASN2 expression under different stresses treatments --- p.65 / Chapter 3.1.3 --- Ammonium assay of wild type seedlings under stress conditions --- p.70 / Chapter 3.2 --- NH4+ regulation on expression of ASN2 promoter --- p.73 / Chapter 3.2.1 --- The cloning ASN2 promoter --- p.73 / Chapter 3.2.1.1 --- Defining of ASN2 promoter region --- p.73 / Chapter 3.2.1.2 --- PCR amplification of ASN2 promoter from genomic sequence --- p.77 / Chapter 3.2.1.3 --- Cloning ASN2 promoter into transient gene expression vector (pBI221 vector) --- p.80 / Chapter 3.2.2 --- Transient gene expression --- p.84 / Chapter 3.2.2.1 --- Arabidopsis leave mesophyll protoplasts isolation --- p.84 / Chapter 3.2.2.2 --- Transformation and GUS expression assay --- p.87 / Chapter 3.3 --- Characterization ASN2 transgenic plants under stress conditions --- p.91 / Chapter 3.3.1 --- Construction of ASN2 transgenic plants --- p.91 / Chapter 3.3.2 --- Characterization of ASN2 transgenic plants --- p.93 / Chapter 3.3.2.1 --- Ammonium assay of ASN2 transgenic plant under different concentration of ammonium --- p.93 / Chapter 3.3.2.2 --- Ammonium assay of ASN2 transgenic plant under high light irradiance --- p.93 / Chapter 3.4 --- Characterization of mutant plants (AzaR) that showed altered ASN2 expression --- p.97 / Chapter 3.4.1 --- Phenotype of azaserine resistant mutant --- p.97 / Chapter 3.4.2 --- ASN2 expression level up-regulated in azaserine resistant mutant --- p.99 / Chapter 3.4.3 --- Checking for linkage between azaserine resistance and ASN2 overexpression --- p.101 / Chapter 3.4.4 --- Crossing the mutant with Landsberg for mapping the azaserine resistant mutant --- p.106 / Chapter 4 --- Discussion --- p.108 / Chapter 4.1 --- ASN2 may relate to ammonium metabolism --- p.108 / Chapter 4.2 --- ASN2 transgenic plants and their response under stresses conditions --- p.111 / Chapter 4.3 --- ASN2 promoter studies by transient gene expression method --- p.112 / Chapter 4.3.1 --- Identification of promoter region --- p.113 / Chapter 4.3.2 --- Isolation of protoplasts from Arabidopsis leaf --- p.114 / Chapter 4.3.3 --- Studies of ASN2 promoter transient gene expression in A thaliana protoplasts --- p.114 / Chapter 4.4 --- Azaserine Resistant Mutant --- p.115 / Chapter 4.4.1 --- Overexpression of ASN2 gene in Azaserine resistant mutant and checking for linkage --- p.115 / Chapter 4.4.2 --- Cross of Azaserine Resistant mutants with Lersberg ecotype for mapping --- p.116 / Chapter 5 --- Conclusion and prospective --- p.118 / References --- p.119 / Appendix --- p.140

No description available.

Wong Lee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 152-169). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgements --- p.v / Abbreviations --- p.vi / List of Figures --- p.vii / List of Tables --- p.ix / Table of Contents --- p.xi / Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- GS-GOGAT cycle in plants and bacteria --- p.2 / Chapter 1.2 --- Roles of PII in regulation of glutamine synthetase in E. coli --- p.4 / Chapter 1.2.1 --- Regulation of GS in E. col --- p.4 / Chapter 1.2.2 --- Transcriptional regulation --- p.5 / Chapter 1.2.2.1 --- The glnALG operon / Chapter 1.2.2.2 --- Intracellular signal through PII and Utase-UR / Chapter 1.2.2.3 --- NRI/NRII as two-component system / Chapter 1.2.3 --- Post-translational regulation by adenylylation and deadenylylation --- p.11 / Chapter 1.2.3.1 --- Role of PII in adenylylation/deadenylylation / Chapter 1.2.4 --- Cumulative Feedback Inhibition --- p.15 / Chapter 1.3 --- PII in other bacteria --- p.15 / Chapter 1.4 --- PII in other higher organisms --- p.20 / Chapter 1.5 --- "PII protein is conserved in enteric bacteria, cyanobacteria, archaea, algae and higher plants" --- p.23 / Chapter 1.6 --- Nitrogen assimilation in higher plants --- p.25 / Chapter 1.6.1 --- Nitrogen uptake --- p.25 / Chapter 1.6.2 --- Primary nitrogen assimilation --- p.28 / Chapter 1.6.3 --- Nitrogen transport and interconversions --- p.28 / Chapter 1.6.4 --- Nitrogen flow --- p.29 / Chapter 1.6.5 --- Molecular regulation of nitrogen assimilation and possible roles of PII in plants --- p.30 / Chapter 1.7 --- Hypothesis of this study --- p.33 / Chapter 2. --- Materials and Methods --- p.35 / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Plant materials --- p.35 / Chapter 2.1.2 --- Equipment and facilities used --- p.35 / Chapter 2.1.3 --- Growth media --- p.37 / Chapter 2.1.4 --- Buffers and solutions used in RNA extraction --- p.38 / Chapter 2.1.5 --- Buffers and solutions used in Northern blot analysis --- p.41 / Chapter 2.1.6 --- Molecular reagents and synthetic oligonucleotides used in the preparation of DIG-labeled probes --- p.45 / Chapter 2.1.7 --- Chemicals used in BioRad Protein Assay --- p.48 / Chapter 2.1.8 --- Chemicals and apparatus used in chlorophylls extraction and quantitation --- p.49 / Chapter 2.1.9 --- Buffers and solutions used in the glutamine synthetase enzyme extraction and assay --- p.49 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Plant growth --- p.50 / Chapter 2.2.2 --- RNA extraction --- p.52 / Chapter 2.2.3 --- Northern blot analysis --- p.54 / Chapter 2.2.4 --- Chlorophyll extraction and quantitation --- p.61 / Chapter 2.2.5 --- Root length measurement --- p.61 / Chapter 2.2.6 --- Total glutamine synthetase enzyme assay --- p.61 / Chapter 2.2.7 --- Measurement of total nitrogen in seeds --- p.64 / Chapter 2.2.8 --- Recording growth and development --- p.64 / Chapter 3. --- Results --- p.65 / Chapter 3.1 --- Overexpression ofPII and truncated PII mRNA in transgenic plants --- p.65 / Chapter 3.2 --- General growth characteristics of PII transgenic plants when grown on soil --- p.70 / Chapter 3.3 --- Physiological changes in the PII and truncated PII transgenic lines --- p.72 / Chapter 3.3.1 --- Fresh weight of the young seedlings --- p.73 / Chapter 3.3.2 --- Chlorophyll contents of shoots --- p.75 / Chapter 3.3.3 --- Root lengths --- p.88 / Chapter 3.3.4 --- Carbon and nitrogen status of seeds --- p.94 / Chapter 3.4 --- Expression of nitrogen assimilatory genes in PII and truncated PII transgenic lines --- p.96 / Chapter 3.4.1 --- Nitrate reductases --- p.96 / Chapter 3.4.2 --- Glutamine synthetases --- p.99 / Chapter 3.4.3 --- Asparagine synthetases --- p.107 / Chapter 3.5 --- Total glutamine synthetase enzyme activity --- p.117 / Chapter 4. --- Discussion --- p.126 / Chapter 4.1 --- Overexpressing PII and truncated PII in the transgenic plants --- p.126 / Chapter 4.2 --- The overall growth and development --- p.135 / Chapter 4.3 --- Chlorophyll --- p.135 / Chapter 4.4 --- Root length --- p.137 / Chapter 4.5 --- Expression of nitrogen assimilatory genes --- p.138 / Chapter 4.5.1 --- Genes encoding nitrate reductase --- p.138 / Chapter 4.5.2 --- Genes encoding glutamine synthetase --- p.140 / Chapter 4.5.3 --- Genes encoding asparagine synthetase --- p.141 / Chapter 4.6 --- Overall GS enzyme levels in the rosette leaves --- p.144 / Chapter 4.7 --- N/C ratio of the seed storage --- p.146 / Chapter 4.8 --- Proposed model for the roles of PII --- p.147 / Chapter 4.9 --- Conclusions --- p.149 / Chapter 4.10 --- Further studies --- p.150 / References --- p.152

Intercellular signalling is essential for multicellular organisms to coordinate growth and development, and is mediated by a huge variety of proteins. Some signalling pathways rely on the proteolytic cleavage of membrane proteins by a relatively newly discovered process of regulated intramembrane proteolysis (RIP), the cleavage of proteins within a transmembrane domain. There are four classes of intramembrane cleaving proteases (ICliPs) – Rhomboids, Site-2-proteases, Signal peptide peptidases and γ-secretase. Of all the ICliPs studied to date, γ-secretase is unique, as it is comprised of a four-protein complex, and is only found in multicellular organisms. A vast amount of research is carried out on the γ-secretase complex, not just because of its role in developmentally important pathways, such as NOTCH signalling, but also due to its role in Alzheimer’s disease. The β-amyloid precursor protein (APP) is cleaved by γ-secretase, and defects in this process result in the release of abnormal peptides that form the senile plaques in the brains of Alzheimer’s disease patients. Homologues of the four components of γ-secretase (PRESENILIN (PS), NICASTRIN (NCT), ANTERIOR PHARYNX DEFECTIVE-1 (APH-1) and PRESENILIN ENHANCER-2 (PEN-2)) are found in plants. The aim of this thesis was to characterise the potential γ-secretase components in Arabidopsis thaliana, to determine whether they form a complex, and to analyse what role, if any, they play in plant signalling. The members of the putative Arabidopsis γ-secretase complex (AtPS1 and 2, AtNCT, AtAPH1 and AtPEN2) were identified through BLAST searches, and found to be uniformly expressed. Analysis of T-DNA insertion mutants in each of these genes, and combinations there of, revealed no gross morphological differences to wild type under normal growth conditions and when subjected to a range of stresses. Protein fusions to GFP under the control of the 35S promoter were constructed and stably transformed into plants. AtPEN2:GFP is expressed throughout the plant, and accumulates in BFA sensitive Golgi bodies in roots. AtPS1:GFP, only accumulates strongly in developing seeds. Native blue PAGE was used to look for high molecular weight complexes (HMW) containing AtPEN2:GFP and AtPS1:GFP. Both fusion proteins were found in similar sized HMW complexes. A variety of methods were used to look for substrates of the iv putative γ-secretase complex in Arabidopsis, and although no specific substrates were identified, a potential role in seed development has been established.

The use of Arabidopsis thaliana populations to understand the genetic basis for natural variation has been highlighted in recent years. The role of adaptation in natural variation remains of key interest. Here, natural variation in growth rate, flowering time and seed production were examined in local populations of A. thaliana from the Edinburgh area using a common garden approach. Growth rate and seed production were found to be highly genetically determined and sometimes correlated, and some genotypes were found to perform consistently better as winter annuals and others as summer annuals, suggesting that adaptation to different seasons might maintain natural variation locally. In order to dissect the environmental factors that could affect growth, these genotypes were also grown under controlled conditions. Photoperiod and temperature were identified as two of the seasonal variables to which different genotypes may be adapted. The relationship between growth rate and competition was also examined. In general, competition exaggerated the differences in performance between genotypes, although the identity of neighbours was observed to have an effect on both growth rate and fitness of A. thaliana in competition. To understand the genetic basis of growth rate variation, the genetic relationships between local populations was examined. Local accessions were usually found to be more closely related to each other than to world-wide accessions, suggesting that their variation did not reflect recent immigration. To examine the genetic architecture of growth rate variation, hybrids between local genotypes with different growth rates were used in QTL analysis. Four chromosomal regions were detected; these regions represent potential growth-rate associated QTL.

Philosophiae Doctor - PhD / Plants are continuously exposed to varying conditions in their environment, to which they have to adapt by manipulating various cellular processes. Environmental (abiotic) and pathogen (biotic) stress are challenges against which plants have to defend themselves. Many plant responses to stress stimuli are a result of cellular processes that can be divided into three sequential steps; namely signal perception, signal transduction m1d execution of a response. Stress signal perception is, in most of these cases, facilitated by cell surface or intracellular receptors that act to recognize molecules presented to the cell. In several cases, hormones are synthesized in response to stress signals and in turn these hormones are perceived by cellular receptors that trigger signal transduction cascades. Propagation of signal transduction cascades is a complex process that results from activation of various signaling molecules within the cell. Second messengers like calcium (Ca2+) and guanosine 3', 5'-cyclic monophosphate (cGMP) play a vital role in mediating many signal transduction processes. The result of these signal transduction cascades is, in most instances, expression of genes that contribute to the plant's ability to cope with the challenges presented to it. Plant natriuretic peptides (PNPs) are novel plant hormones that regulate water and salt homeostasis via cGMP-dependent signaling pathways that involve deployment of Ca2+. The aim of this study is to partially characterize a PNP and a guanylyl cyclase, both from Arabidopsis thaliana. Guanylyl cyclases synthesize cGMP from the hydrolysis of guanosine 5' -triphosphate (GTP) in the cell. The study also aims to investigate the effect of drought and salinity on cGMP levels in plants, using sorbitol to mimic the osmolarity/dehydration effect of drought and NaCl as a source of salinity stress and thus link NaCl and sorbitol responses to both AtPNP-A and cGMP up-regulation.

Li, Man Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 109-119). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Abstract --- p.III / 摘要 --- p.V / Acknowledgements --- p.VI / Abbreviations --- p.VIII / Abbreviations of Chemicals --- p.X / List of Tables --- p.XI / List of Figures --- p.XII / Table of Contents --- p.XIII / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- General amino acid control in yeast --- p.1 / Chapter 1.2 --- Mammalian eIF2α kinases --- p.7 / Chapter 1.2.1 --- Heme-regulated inhibitor kinase (EIF2AK1/HRI) --- p.7 / Chapter 1.2.2 --- Protein kinase dsRNA-dependent (EIF2AK2/PKR) --- p.8 / Chapter 1.2.3 --- PKR-like ER kinase (EIF2AK3/PERK) --- p.9 / Chapter 1.2.4 --- General control non-repressible 2 (EIF2AK4/GCN2) --- p.10 / Chapter 1.2.5 --- Activating transcription factor 4 (ATF4) --- p.11 / Chapter 1.3 --- Plant General Amino Acid Control --- p.12 / Chapter 1.3.1 --- Studies of the homolog of GCN2 in Arabidopsis thaliana --- p.12 / Chapter 1.3.2 --- Studies of the homolog of other eIF2a kinase in plant --- p.14 / Chapter 1.3.3 --- Studies of the homolog of other GAAC components --- p.14 / Chapter 1.4 --- Previous works in our lab --- p.15 / Chapter 1.5 --- Hypothesis and Objectives --- p.17 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.18 / Chapter 2.1.1 --- "Bacterial cultures, plant materials and vectors" --- p.18 / Chapter 2.1.2 --- Primers --- p.21 / Chapter 2.1.3 --- Commercial kits --- p.25 / Chapter 2.1.4 --- "Buffer, solution, gel and medium" --- p.25 / Chapter 2.1.5 --- "Chemicals, reagents and consumables" --- p.25 / Chapter 2.1.6 --- Enzymes --- p.25 / Chapter 2.1.7 --- Antibodies --- p.25 / Chapter 2.1.8 --- Equipments and facilities --- p.25 / Chapter 2.2 --- Methods --- p.26 / Chapter 2.2.1 --- Growth conditions of Arabidopsis thaliana --- p.26 / Chapter 2.2.1.1 --- Surface sterilize of Arabidopsis thaliana seed --- p.26 / Chapter 2.2.1.2 --- Growing of Arabidopsis thaliana --- p.26 / Chapter 2.2.1.3 --- Treatment of Arabidopsis seedling --- p.26 / Chapter 2.2.2 --- Basic molecular techniques --- p.27 / Chapter 2.2.2.1 --- Liquid culture of Escherichia coli --- p.27 / Chapter 2.2.2.2 --- Preparation of plasmid DNA --- p.27 / Chapter 2.2.2.3 --- Restriction digestion --- p.27 / Chapter 2.2.2.4 --- DNA purification --- p.28 / Chapter 2.2.2.5 --- DNA gel electrophoresis --- p.28 / Chapter 2.2.2.6 --- DNA ligation --- p.29 / Chapter 2.2.2.7 --- CaCl2 mediated E. coli transformation --- p.29 / Chapter 2.2.2.8 --- Preparation of DNA fragment for cloning --- p.29 / Chapter 2.2.2.9 --- PCR reaction for screening positive E. coli transformants --- p.30 / Chapter 2.2.2.10 --- DNA sequencing --- p.30 / Chapter 2.2.2.11 --- RNA extraction from plant tissue with tRNA --- p.31 / Chapter 2.2.2.12 --- Extraction of RNA without tRNA --- p.31 / Chapter 2.2.2.13 --- cDNA synthesis --- p.32 / Chapter 2.2.2.14 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.33 / Chapter 2.2.2.15 --- Western blotting --- p.33 / Chapter 2.2.3 --- Sub-cloning of AtGCN2 --- p.34 / Chapter 2.2.3.1 --- Sub-cloning full length AtGCN2 into pMAL-c2 --- p.36 / Chapter 2.2.3.2 --- Sub-cloning of the N-terminal sequence of AtGCN2 into pMAL-c2 --- p.38 / Chapter 2.2.3.3 --- Sub-cloning of the C-terminal sequence of AtGCN2 into pMAL-c2 --- p.38 / Chapter 2.2.4 --- Cloning of the eIF2α candidates for the in vitro assay --- p.41 / Chapter 2.2.4.1 --- Cloning of At2g40290 (putative eIF2α candidate) --- p.41 / Chapter 2.2.4.2 --- Cloning of At5g05470 (putative eIF2α candidate) into pBlueScript KS II + --- p.43 / Chapter 2.2.4.3 --- Sub-cloning of At5g05470 into pGEX-4T-1 --- p.43 / Chapter 2.2.4 --- Expression and purification of fusion proteins --- p.45 / Chapter 2.2.5 --- Expression of fusion proteins in E. coli --- p.45 / Chapter 2.2.5.2 --- Extraction of E. coli soluble proteins --- p.45 / Chapter 2.2.5.3 --- Purification of GST tagged fusion protein --- p.46 / Chapter 2.2.5.4 --- Purification of MBP tagged fusion protein --- p.46 / Chapter 2.2.5.5 --- Concentration of purified fusion proteins --- p.46 / Chapter 2.2.5.6 --- MS/MS verification of purified fusion proteins --- p.47 / Chapter 2.2.6 --- Gel mobility shift assay --- p.47 / Chapter 2.2.6.1 --- Synthesis of short biotinylated RNA --- p.47 / Chapter 2.2.6.2 --- Ligation of short biotinylated RNA with tRNA --- p.48 / Chapter 2.2.6.3 --- Gel mobility shift assay --- p.48 / Chapter 2.2.6.4 --- Blotting of the sample on to nitrocellulose membrane --- p.48 / Chapter 2.2.6.5 --- Detection of the tRNA on the membrane --- p.49 / Chapter 2.2.6.6 --- Detection of the MBP fusion proteins on the membrane --- p.49 / Chapter 2.2.7 --- In vitro kinase assay of AtGCN2 --- p.49 / Chapter 2.2.8 --- In vitro translation inhibition assay --- p.50 / Chapter 2.2.8.1 --- In vitro transcription of HA mRNA --- p.50 / Chapter 2.2.8.2 --- In vitro translation --- p.51 / Chapter 2.2.8.3 --- Detection of the protein dot blot --- p.51 / Chapter 2.2.9 --- Gene expression analysis by real time PCR --- p.52 / Chapter 2.2.10 --- Total seed nitrogen analysis --- p.53 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Blast search results suggested that AtGCN2 may be the sole eIF2α kinase in Arabidopsis thaliana --- p.54 / Chapter 3.2 --- Existence of two eIF2α candidates in Arabidopsis thaliana genome --- p.59 / Chapter 3.3 --- Fusion proteins were successfully expressed and purified --- p.63 / Chapter 3.4 --- C-terminal of AtGCN2 has a higher affinity toward tRNA than rRNA --- p.67 / Chapter 3.5 --- Both eIF2α candidates can be phosphorylated by full length AtGCN2 in vitro --- p.70 / Chapter 3.6 --- AtGCN2 can inhibit translation in vitro --- p.72 / Chapter 3.7 --- Overexpression of AtGCN2 did not affect expression of selected genes --- p.74 / Chapter 3.8 --- Overexpression of AtGCN2 did not affect seed nitrogen content and C:N ratio under normal growth conditions --- p.83 / Chapter Chapter 4 --- Discussion --- p.85 / Chapter 4.1 --- Existing evidence supported that AtGCN2 is the sole eIF2α kinase in Arabidopsis thaliana --- p.85 / Chapter 4.2 --- Kinase activities of AtGCN2 and its two substrates in Arabidopsis --- p.86 / Chapter 4.3 --- C-terminal binds tRNA in the gel mobility shift assay --- p.88 / Chapter 4.4 --- Overexpression of AtGCN2 did not affect gene expression of the transgenic lines under nitrogen starvation and azerserine treatment --- p.90 / Chapter 4.5 --- Overexpression of AtGCN2 did not alter the seed nitrogen content --- p.91 / Chapter 4.6 --- Existence of GCN4 and ATF4 in plant --- p.92 / Chapter 4.7 --- Alternative model without GCN4 and ATF4 homolog --- p.93 / Chapter 4.8 --- Possible application of the in vitro kinase assay --- p.94 / Chapter 4.9 --- Possible application of the in vitro translation inhibition analysis platform in future study --- p.95 / Chapter Chapter 5 --- Conclusion and Future Prospective --- p.97 / Appendices / Appendix I Commercial kits used in this project --- p.98 / "Appendix II Buffer, solution, gel and medium" --- p.99 / "Appendix III Chemicals, reagents and consumables" --- p.102 / Appendix IV Enzymes --- p.103 / Appendix V Antibodies --- p.104 / Appendix VI Equipments and facilities --- p.105 / Appendix VII Supplementary Data --- p.106 / Appendix VIII Amplification efficiency of real time primers --- p.108 / References --- p.109

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No description available.