Plant as bioreactor: transgenic expression of malaria surface antigen in plants.

by Ng Wang Kit.Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.Includes bibliographical references (leaves 131-139).Abstracts in English and Chinese.Acknowledgements --- p.iiiAbstract --- p.vList of Tables --- p.ixList of Figures --- p.xList of Abbreviations --- p.xiiiTable of Contents --- p.xvChapter Chapter 1: --- General Introduction --- p.1Chapter Chapter 2: --- Literature Review --- p.3Chapter 2.1 --- Malaria --- p.3Chapter 2.1.1 --- Global picture --- p.3Chapter 2.1.2 --- Malaria mechanics --- p.4Chapter 2.1.3 --- Life cycle of malaria parasite --- p.4Chapter 2.2 --- Treatment of malaria ´ؤ malaria drugs --- p.5Chapter 2.2.1 --- Antimalarial drugs --- p.5Chapter 2.2.2 --- Drug resistance --- p.6Chapter 2.3 --- Treatment of malaria - malarial vaccines --- p.7Chapter 2.3.1 --- Malarial vaccine developments --- p.7Chapter 2.3.2 --- Transmission blocking vaccines --- p.7Chapter 2.3.3 --- Pre-erythrocytic vaccines --- p.9Chapter 2.3.4 --- Blood stage vaccines --- p.10Chapter 2.4 --- The major merozoite protein - gpl95 --- p.11Chapter 2.5 --- Plants as bioreactors --- p.12Chapter 2.5.1 --- Products of transgenic plants --- p.13Chapter 2.6 --- Transgenic plants for production of subunit vaccines --- p.14Chapter 2.6.1 --- Norwalk virus capsid protein production --- p.15Chapter 2.6.2 --- Hepatitis B surface antigen production --- p.15Chapter 2.7 --- Tobacco and Arabidopsis as model plants --- p.16Chapter 2.7.1 --- Arabidopsis --- p.16Chapter 2.7.2 --- Tobacco --- p.17Chapter 2.8 --- Transformation methods --- p.17Chapter 2.8.1 --- Direct DNA uptake --- p.17Chapter 2.8.1.1 --- Plant protoplast transformation --- p.17Chapter 2.8.1.2 --- Biolistic transformation --- p.18Chapter 2.8.2 --- Agrobacterium-mediated transformation --- p.18Chapter 2.8.2.1 --- Leaf-disc technique --- p.18Chapter 2.8.2.2 --- In planta transformation --- p.19Chapter 2.9 --- Phaseolin --- p.20Chapter 2.10 --- Detection and purification of recombinant products - Histidine tag --- p.21Chapter 2.11 --- Aims of study and hypotheses --- p.22Chapter Chapter 3: --- Materials and Methods --- p.24Chapter 3.1 --- Introduction --- p.24Chapter 3.2 --- Chemicals --- p.24Chapter 3.3 --- Expression in tobacco system --- p.24Chapter 3.3.1 --- Plant materials --- p.24Chapter 3.3.2 --- Bacterial strains --- p.25Chapter 3.3.3 --- Chimeric gene construction for tobacco transformation --- p.25Chapter 3.3.3.1 --- The cloning of pTZPhasp/flgp42-His/Phast (F1) --- p.26Chapter 3.3.3.2 --- The cloning of pBKPhasp-sp/flgp42-His/Phast (P9) --- p.30Chapter 3.3.3.3 --- The cloning of pHM2Ubip/flgp42-His/Nost (C2) --- p.30Chapter 3.3.4 --- Confirmation of sequence fidelity of chimeric gene by DNA sequencing --- p.33Chapter 3.3.5 --- Cloning of chimeric gene into binary vector --- p.34Chapter 3.3.6 --- Triparental mating of Agrobacterium tumefaciens LBA4404/pAL4404 --- p.35Chapter 3.3.7 --- Tobacco transformation and regeneration --- p.36Chapter 3.3.8 --- GUS assay --- p.37Chapter 3.3.9 --- Genomic DNA isolation --- p.37Chapter 3.3.10 --- PCR amplification and detection of transgene --- p.38Chapter 3.3.11 --- Southern blot analysis --- p.38Chapter 3.3.12 --- Total seeds RNA isolation --- p.39Chapter 3.3.13 --- RT-PCR --- p.39Chapter 3.3.14 --- Northern blot analysis --- p.40Chapter 3.3.15 --- Protein extraction and SDS-PAGE --- p.40Chapter 3.3.16 --- Western blot analysis --- p.41Chapter 3.4 --- Expression in Arabidopsis system --- p.42Chapter 3.4.1 --- Plant materials --- p.42Chapter 3.4.2 --- Bacterial strains --- p.42Chapter 3.4.3 --- Chimeric gene construction --- p.42Chapter 3.4.3.1 --- The cloning of pBKPhasp-sp/His/EK/p42/Phast (DH) --- p.43Chapter 3.4.3.2 --- The cloning of pTZPhaSp/His/EK/p42/Phast (EH) --- p.45Chapter 3.4.3.3 --- The cloning of pBKPhasp-sp/His/EK/flgp42/Phast (DHF) and pTZPhasp/His/EK/flgp42/Phast (EHF) --- p.45Chapter 3.4.4 --- Confirmation of sequence fidelity of chimeric genes --- p.45Chapter 3.4.5 --- Cloning of chimeric gene into Agrobacterium binary vector --- p.49Chapter 3.4.6 --- Transformation of Agrobacterium tumefaciens GV3101/pMP90 with chimeric gene constructs --- p.49Chapter 3.4.7 --- Arabidopsis Transformation --- p.49Chapter 3.4.8 --- Vacuum infiltration transformation --- p.50Chapter 3.4.9 --- Selection of successful transformants --- p.51Chapter 3.4.10 --- Selection for homozygous plants with single gene insertion --- p.51Chapter 3.4.11 --- GUS assay --- p.52Chapter 3.4.12 --- Genomic DNA isolation --- p.52Chapter 3.4.13 --- PCR amplification and detection of transgenes --- p.52Chapter 3.4.14 --- Southern Blot analysis --- p.52Chapter 3.4.15 --- Total siliques RNA isolation --- p.53Chapter 3.4.16 --- RT-PCR --- p.53Chapter 3.4.17 --- Northern blot analysis --- p.53Chapter 3.4.17 --- Protein extraction and SDS-PAGE --- p.54Chapter 3.4.18 --- Western blot analysis --- p.54Chapter 3.5 --- In vitro transcription and translation --- p.54Chapter 3.5.1 --- In vitro transcription --- p.54Chapter 3.5.2 --- In vitro translation --- p.55Chapter 3.6 --- Particle bombardment of GUS fusion gene --- p.56Chapter 3.6.1 --- Chimeric gene constructs --- p.56Chapter 3.6.2 --- Particle bombardment using snow bean cotyledon --- p.61Chapter Chapter 4: --- Results --- p.63Chapter 4.1 --- Tobacco system --- p.63Chapter 4.1.1 --- Chimeric gene constructs --- p.63Chapter 4.1.2 --- Tobacco transformation and regeneration --- p.65Chapter 4.1.3 --- GUS activity assay --- p.67Chapter 4.1.4 --- Molecular analysis of transgene integration --- p.68Chapter 4.1.4.1 --- Genomic DNA extraction and PCR --- p.68Chapter 4.1.4.2 --- Southern blot analysis --- p.70Chapter 4.1.5 --- Molecular analysis of transgene expression --- p.72Chapter 4.1.5.1 --- Total RNA isolation and RT-PCR --- p.72Chapter 4.1.5.2 --- Northern blot analysis --- p.75Chapter 4.1.6 --- Genomic PCR to confirm whole gene transfer --- p.76Chapter 4.1.7 --- Biochemical analysis of transgene expression --- p.78Chapter 4.1.7.1 --- Protein extraction and SDS-PAGE --- p.78Chapter 4.1.7.2 --- Western blot analysis --- p.78Chapter 4.2 --- Arabidopsis system --- p.83Chapter 4.2.1 --- Chimeric gene constructs --- p.83Chapter 4.2.2 --- Arabidopsis transformation and selection --- p.85Chapter 4.2.3 --- Selection of transgenic plants --- p.87Chapter 4.2.4 --- Assay of GUS activity --- p.91Chapter 4.2.5 --- Molecular analysis of transgene integration --- p.92Chapter 4.2.5.1 --- Genomic DNA extraction and PCR --- p.92Chapter 4.2.5.2 --- Southern blot analysis --- p.96Chapter 4.2.6 --- Molecular analysis of transgene expression --- p.99Chapter 4.2.6.1 --- Total RNA isolation and RT-PCR --- p.99Chapter 4.2.6.2 --- Northern blot analysis --- p.106Chapter 4.2.7 --- Genomic PCR for confirmation of whole gene transfer --- p.107Chapter 4.2.8 --- Biochemical analysis of transgene expression --- p.108Chapter 4.2.8.1 --- Protein extraction and SDS-PAGE --- p.108Chapter 4.2.8.2 --- Western blot analysis --- p.108Chapter 4.3 --- In vitro transcription and translation --- p.112Chapter 4.4 --- Particle bombardment of p42/ GUS fusion gene --- p.115Chapter Chapter 5: --- Discussion and Future perspectives --- p.117Chapter 5.1 --- Failure in detecting transgene expression --- p.117Chapter 5.2 --- Poor transgene expression --- p.120Chapter 5.2.1 --- Bacillus thuringiensis toxin and green fluorescent protein --- p.120Chapter 5.2.2 --- AT-richness --- p.121Chapter 5.2.3 --- Deleterious sequence - AUUUA --- p.123Chapter 5.2.4 --- Presence of AAUAAA or AAUAAA-like motifs --- p.125Chapter 5.2.5 --- Codon usage --- p.126Chapter 5.3 --- Future perspectives --- p.127Chapter Chapter 6: --- Conclusion --- p.129References --- p.13