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

• Other Authors: Cheng, Man Kin.
• Format: Others
• Language: English; Chinese
• Published: 1999
• Subjects: Arabidopsis > Genetics; Lysine; Transgenic plants; Genetic transformation; Gene expression
• Online Access: http://library.cuhk.edu.hk/record=b5890085; http://repository.lib.cuhk.edu.hk/en/item/cuhk-322630

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