Transgenic expression of a chimeric gene encoding a lysine-rich protein in arabidopsis.

by Cheng Man Kin.Thesis (M.Phil.)--Chinese University of Hong Kong, 1999.Includes bibliographical references (leaves 71-76).Abstracts in English and Chinese.Thesis committee --- p.iAbstract --- p.iiAcknowledgements --- p.ivAbbreviations --- p.vTable of contents --- p.viiList of figures --- p.xList of tables --- p.xiChapter Chapter 1: --- General introduction --- p.1Chapter Chapter 2: --- Literature review --- p.3Chapter 2.1 --- Nutritional quality of plant proteins --- p.3Chapter 2.2 --- Using traditional plant breeding method to enhance amino acid quality of plant proteins --- p.3Chapter 2.3 --- Molecular strategies to enhance amino acid quality of plant proteins --- p.4Chapter 2.3.1 --- Heterologous gene expression --- p.5Chapter 2.3.2 --- Protein sequence modification --- p.8Chapter 2.3.3 --- Modification of biosynthesis pathway --- p.10Chapter 2.3.4 --- Synthetic gene expression --- p.11Chapter 2.3.5 --- Homologous gene overexpression --- p.13Chapter 2.4 --- Arabidopsis --- p.14Chapter 2.4.1 --- Arabidopsis as a model plant --- p.14Chapter 2.4.2 --- Transformation methods --- p.14Chapter 2.4.2.1 --- Direct DNA uptake --- p.15Chapter 2.4.2.2 --- Agrobacterium-mediated transformation --- p.15Chapter 2.5 --- Winged Bean Lysine-Rich protein --- p.17Chapter 2.5.1 --- Identification of winged bean polypeptides rich in lysine --- p.17Chapter 2.5.2 --- Cloning of the lysine-rich protein gene --- p.17Chapter 2.5.3 --- Further characterization of the WBLRP gene --- p.18Chapter 2.6 --- Phaseolin --- p.19Chapter Chapter 3: --- Expression of LRP in transgenic Arabidopsis --- p.20Chapter 3.1 --- Introduction --- p.20Chapter 3.2 --- Materials and methods --- p.21Chapter 3.2.1 --- Targeting LRP to cytosol --- p.21Chapter 3.2.1.1 --- Chemicals --- p.21Chapter 3.2.1.2 --- Plant materials --- p.21Chapter 3.2.1.3 --- Bacterial strains --- p.22Chapter 3.2.1.4 --- Construction of chimeric LRP gene (pBILRP-1) --- p.22Chapter 3.2.1.4.1 --- PCR amplification of LRP --- p.22Chapter 3.2.1.4.2 --- Cloning of PCR-amplified LRP into vector pD3-8 --- p.26Chapter 3.2.1.4.3 --- Cloning of recombinant plasmid pLRP-1 into binary vector --- p.26Chapter 3.2.1.5 --- Transformation of Agrobacterium with pBILRP-1 --- p.27Chapter 3.2.1.6 --- Vacuum infiltration transformation of Arabidopsis --- p.28Chapter 3.2.1.7 --- Selection of transgenic plants --- p.29Chapter 3.2.1.8 --- GUS assay --- p.30Chapter 3.2.1.9 --- DNA isolation --- p.31Chapter 3.2.1.10 --- PCR amplification and detection of transgenes --- p.31Chapter 3.2.1.11 --- Southern blot hybridization --- p.31Chapter 3.2.1.12 --- RNA isolation --- p.32Chapter 3.2.1.13 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.32Chapter 3.2.1.14 --- Protein extraction and SDS-PAGE --- p.33Chapter 3.2.1.15 --- Protein sequencing --- p.33Chapter 3.2.1.16 --- Amino acid analysis --- p.34Chapter 3.2.2 --- Targeting LRP to protein bodies --- p.35Chapter 3.2.2.1 --- Chemicals --- p.35Chapter 3.2.2.2 --- Plant materials --- p.35Chapter 3.2.2.3 --- Bacterial strains --- p.35Chapter 3.2.2.4 --- Construction of chimeric LRP gene (pBILRP-2) --- p.35Chapter 3.2.2.4.1 --- Site-directed mutagenesis --- p.36Chapter 3.2.2.4.2 --- Cloning of the mutated phaseolin fragment into pBluescript --- p.36Chapter 3.2.2.4.3 --- PCR amplification of LRP --- p.39Chapter 3.2.2.4.4 --- Insertion of LRP into plasmid pBK/phas* --- p.39Chapter 3.2.2.4.5 --- Insertion of plasmid pLRP-2 into Agrobacterium binary vector --- p.41Chapter 3.2.2.5 --- Transformation of Agrobacterium with pBILRP-2 --- p.41Chapter 3.2.2.6 --- Vacuum infiltration transformation of Arabidopsis --- p.41Chapter 3.2.2.7 --- Selection of transgenic plants --- p.41Chapter 3.3 --- Results and discussion --- p.42Chapter 3.3.1 --- Targeting LRP to protein bodies --- p.42Chapter 3.3.1.1 --- Morphology of transgenic Arabidopsis --- p.42Chapter 3.3.1.2 --- Selection of transgenic plants --- p.42Chapter 3.3.2 --- Targeting LRP to cytosol --- p.46Chapter 3.3.2.1 --- Morphology of transgenic Arabidopsis --- p.46Chapter 3.3.2.2 --- Selection of transgenic plants --- p.46Chapter 3.3.2.3 --- Detection of GUS activity --- p.49Chapter 3.3.2.4 --- Integration of LRP transgene into Arabidopsis genome --- p.54Chapter 3.3.2.5 --- LRP transcript in transgenic Arabidopsis --- p.58Chapter 3.3.2.6 --- Stable accumulation of LRP in transgenic Arabidopsis --- p.61Chapter 3.3.2.7 --- Amino acid analysis of seed protein --- p.64Chapter Chapter 4: --- General discussion --- p.67Conclusion --- p.70References --- p.7