Molecular studies on sweet protein mabinlin: thermal stability.

Leung Chun-wah.Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.Includes bibliographical references (leaves 113-122).Abstracts in English and Chinese.Thesis committee --- p.iStatement --- p.iiAcknowledgment --- p.iiiAbstract --- p.vTable of contents --- p.ixList of abbreviations --- p.xivList of figures --- p.xviiList of tables --- p.xixChapter 1 --- LITERATURE REVIEW --- p.1Chapter 1.1 --- Introduction --- p.1Chapter 1.2 --- Artificial sweeteners --- p.3Chapter 1.2.1 --- SACCHARIN --- p.3Chapter 1.2.2 --- cyclamate --- p.4Chapter 1.2.3 --- Aspartame --- p.4Chapter 1.2.4 --- acesulfame-k --- p.5Chapter 1.2.5 --- SUCRALOSE --- p.5Chapter 1.3 --- natural sweet plant proteins --- p.7Chapter 1.3.1 --- THAUMATIN --- p.7Chapter 1.3.2 --- MONELLIN --- p.10Chapter 1.3.3 --- CURCULIN --- p.11Chapter 1.3.4 --- PENTADIN AND BRAZZEIN --- p.11Chapter 1.3.5 --- MIRACULIN --- p.12Chapter 1.3.6 --- MABINLIN --- p.12Chapter 1.4 --- Genetic Engineering of Sweet Plant Protein --- p.19Chapter 1.4.1 --- biotechnological studies on thaumatin --- p.20Chapter 1.4.1.1 --- Protein modification and sweetness --- p.20Chapter 1.4.1.2 --- Transgenic expression in microbes --- p.21Chapter 1.4.1.3 --- Transgenic expression in higher plants --- p.23Chapter 1.4.2 --- BIOTECHNOLOGICAL STUDIES ON MONELLIN --- p.24Chapter 1.4.2.1 --- Gene modification and transgenic expression in microbes --- p.24Chapter 1.4.2.2 --- Transgenic expression in plants --- p.25Chapter 1.4.3 --- TRANSGENIC EXPRESSION OF MABINLIN IN PLANTS --- p.26Chapter 1.5 --- phaseolin and its regulatory sequences --- p.27Chapter 1.6 --- ARABIDOPSIS --- p.29Chapter 1.6.1 --- ARABIDOPSIS THALIANA as a model plant --- p.29Chapter 1.6.2 --- Transformation methods --- p.29Chapter 1.6.2.1 --- Direct DNA uptake --- p.30Chapter 1.6.2.2 --- Agrobacterium-mediated transformation --- p.31Chapter 1.6.2.3 --- In planta transformation --- p.31Chapter 2 --- GENKRAL INTRODUTION AND HYPOTHESIS --- p.22Chapter 2.1 --- General Introduction --- p.33Chapter 2.2 --- Hypothesis --- p.34Chapter 3 --- MOLECULAR STUDIES ON SWEET PROTEIN MARINLIN : THERMAL STABILITY --- p.28Chapter 3.1 --- Introduction --- p.38Chapter 3.2 --- Materials --- p.40Chapter 3.2.1 --- laboratory wares --- p.40Chapter 3.2.2 --- Equipments --- p.40Chapter 3.2.3 --- Chemicals --- p.40Chapter 3.2.4 --- commerical kits --- p.41Chapter 3.2.5 --- DNA primers --- p.42Chapter 3.2.6 --- DNA plasmids --- p.43Chapter 3.2.7 --- bacterial strains --- p.43Chapter 3.2.8 --- Plant materials --- p.44Chapter 3.2.9 --- Protein and Antibody --- p.44Chapter 3.3 --- Methods --- p.45Chapter 3.3.1 --- Transformation of Arabidopsis with mbliii and mbli genes --- p.45Chapter 3.3.1.1 --- Construction of mutant MBLIII and MBLI genes containing single codon mutation by megaprimer PCR --- p.45Chapter 3.3.1.2 --- Cloning of PCR-amplified MBLIII and MBLI cDNAs into vector pD3-8 --- p.48Chapter 3.3.1.3 --- In vitro site-directed mutagensis (for the construction of MBLIII and MBLI cDNAs containing single codon mutation) --- p.49Chapter 3.3.1.4 --- Cloning of the wild-type and mutated MBLIII and MBLI cDNA into vector pTZ / phas --- p.53Chapter 3.3.1.5 --- Confirmation of sequence fidelity and mutated codon in MBLIII and MBLI cDNA by DNA sequencing --- p.53Chapter 3.3.1.6 --- Transfer of wild-type MBLIII and MBLI cDNA flanked by phaseolin regulatory sequence into Agrobacterium binary vector --- p.55Chapter 3.3.1.7 --- Transformation of Agrobacterium with pBI / phas / MBLIII and pBI / phas / MBLI chimeric gene constructs --- p.57Chapter 3.3.1.8 --- Vacuum infiltration transformation of A rabidopsis --- p.58Chapter 3.3.1.9 --- Screening of homozygous transgenic Arabidopsis --- p.59Chapter 3.3.2 --- Expression analysis of MBLIII transgene --- p.61Chapter 3.3.2.1 --- GUS assay of transgenic plants --- p.61Chapter 3.3.2.2 --- Genomic DNA isolation from transgenic plants --- p.61Chapter 3.3.2.3 --- PCR amplification of transgene --- p.62Chapter 3.3.2.4 --- Total RNA isolation from transgenic Arabidopsis --- p.63Chapter 3.3.2.5 --- RT-PCR of total RNA from transgenic Arabidopsis --- p.64Chapter 3.3.2.6 --- Verification of the presence of mutagenic site and the fidelity of RNA transcript from transgenic Arabidopsis --- p.65Chapter 3.3.2.7 --- Protein extraction and tricine SDS-PAGE of putative transgenic protein from Arabidopsis --- p.65Chapter 3.3.2.8 --- N-terminal amino acid sequencing --- p.66Chapter 3.3.2.9 --- Isoelectric precipitation of MBL --- p.67Chapter 3.3.2.10 --- Production of polyclonal antibody against purified MBL --- p.67Chapter 3.3.2.11 --- Western-blotting and immunodectection of Arabidopsis protein by anti-MBL polyclonal antibody --- p.69Chapter 3.4 --- results & discussion --- p.71Chapter 3.4.1 --- Site-specific mutations of Arginine residue in mbliii cdna and glutamine in mbli cdna --- p.71Chapter 3.4.1.1 --- Megaprimer PCR --- p.71Chapter 3.4.1.2 --- Cloning into the seed-specific expression vector pD38 --- p.74Chapter 3.4.1.3 --- In vitro site-directed mutagenesis --- p.76Chapter 3.4.2 --- Construction of plant expression vectors containing chimeric MBLIII and MBLI --- p.80Chapter 3.4.2.1 --- Cloning of MBLIII and MBLI cDNAs into the seed-specific expression vector pTZ / phas --- p.80Chapter 3.4.2.2 --- Cloning into the plant expression vector pBI121 --- p.83Chapter 3.4.3 --- Generation of homozygous transgenic Arabidopsis --- p.84Chapter 3.4.3.1 --- Screening of transgenic R1 Arabidopsis --- p.84Chapter 3.4.3.2 --- Screening of transgenic R2 plants --- p.86Chapter 3.4.3.3 --- Screening of homozygous R3 transgenic plants --- p.88Chapter 3.4.4 --- Detection of MBLIII transgene in Arabidopsis --- p.89Chapter 3.4.4.1 --- Gus Assay --- p.89Chapter 3.4.4.2 --- Detection of transgene integration --- p.90Chapter 3.4.5 --- DETECTION of MBLIII TRANSCRIPT IN TRANSGENIC arabidopsis --- p.92Chapter 3.4.5.1 --- RT-PCR (Reverse-transcription polymerase chain reaction) --- p.92Chapter 3.4.5.2 --- Verification of the presence of the mutant codon and sequence fidelity of the RT-PCR product --- p.94Chapter 3.4.6 --- DETECTION OF MBL III PROTEIN IN TRANSGENIC arabidopsis --- p.97Chapter 3.4.6.1 --- Expression of MBL protein --- p.97Chapter 3.4.6.2 --- Isoelectric precipitation --- p.101Chapter 3.4.6.3 --- Assay of titers and quality of primary polyclonal antibody against purified MBL protein --- p.103Chapter 3.4.6.4 --- Western blot / Immunodetection --- p.106Chapter 4 --- GENERAL DISCUSSION --- p.109Conclusion --- p.112References --- p.11