Characterization of PII and truncated PII transgenic, Arabidopsis thaliana.

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