Molecular cloning & expression of mannose-binding lectin from Chinese herb, yu chu (Polygonatum odoratum) in rice

by Wai Ching Man.Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.Includes bibliographical references (leaves 154-159).Abstracts in English and Chinese.Statement --- p.iiAcknowledgements --- p.iiiAbstract --- p.v摘要 --- p.viiList of Abbreviations --- p.viiiTable of contents --- p.xList of Tables --- p.xivList of Figures --- p.xvChapter Chapter 1 --- Introduction --- p.1Chapter Chapter 2 --- Literature review --- p.4Chapter 2.1 --- Plant lectins --- p.4Chapter 2.1.1 --- Introduction --- p.4Chapter 2.1.2 --- Definition and subdivision of plant lectins --- p.4Chapter 2.2 --- Monocot mannose-binding lectins --- p.6Chapter 2.2.1 --- Occurrence and carbohydrate binding specificity --- p.6Chapter 2.2.2 --- Molecular structure and amino acid sequence --- p.7Chapter 2.2.3 --- "Molecular cloning, biosynthesis and post-translational modification" --- p.10Chapter 2.2.4 --- Mannose-binding lectins of Family Liliaceae --- p.11Chapter 2.2.4.1 --- Tulipa gesneriana lectins (TGL) --- p.12Chapter 2.2.4.2 --- Aloe arborescens lectins (AAL) --- p.13Chapter 2.2.4.3 --- Polygonatum multiflorum agglutinin (PMA) and lectin-related protein --- p.14Chapter 2.3 --- Polygonatum odoratum lectins (POL) --- p.15Chapter 2.3.1 --- Isolation and purification of POL from Yu Chu --- p.15Chapter 2.3.2 --- Agglutinating activity and anti-viral activities of POL --- p.17Chapter 2.3.3 --- Bacterial expression of POL in Escherichia coli --- p.18Chapter 2.4 --- Plant-based production of recombinant proteins --- p.20Chapter 2.4.1 --- Advantages of using plants as expression system --- p.20Chapter 2.4.2 --- Plant-derived recombinant proteins --- p.22Chapter 2.5 --- Expression of heterologous proteins in rice --- p.24Chapter 2.5.1 --- The facts of rice --- p.24Chapter 2.5.2 --- Rice storage proteins --- p.25Chapter 2.5.2 --- Expression of lysine-rich protein (LRP)/glutelin fusion proteinin rice seeds --- p.28Chapter 2.5.3 --- Expression of Galanthus nivalis agglutinin in rice --- p.29Chapter 2.6 --- Protein trafficking in plants --- p.30Chapter 2.6.1 --- Golgi-dependent pathways --- p.30Chapter 2.6.2 --- Golgi-independent pathway --- p.32Chapter 2.6.3 --- Expression of protein targeting determinants in tobacco plants and suspension cells --- p.33Chapter Chapter 3 --- Materials and Methods --- p.35Chapter 3.1 --- Introduction --- p.35Chapter 3.2 --- Chemcials --- p.35Chapter 3.3 --- Bacterial strains --- p.35Chapter 3.4 --- Cloning of POL cDNA --- p.36Chapter 3.4.1 --- Plant materials --- p.36Chapter 3.4.2 --- RNA extraction --- p.36Chapter 3.4.3 --- RT-PCR amplification of POL cDNA --- p.36Chapter 3.4.4 --- 5'RACE and 3'RACE --- p.38Chapter 3.4.5 --- Sequencing of POL cDNA --- p.39Chapter 3.5 --- Analysis of POL protein --- p.40Chapter 3.5.1 --- Protein extraction and Tricine-SDS PAGE --- p.40Chapter 3.5.2 --- Western blot analysis --- p.41Chapter 3.6 --- Chimeric gene construction --- p.42Chapter 3.6.1 --- Construction of the Cauliflower mosaic virus (CaMV)35S promoter/POL constructs --- p.44Chapter 3.6.2 --- Construction of the glutelin-1 promoter/POL constructs --- p.48Chapter 3.6.3 --- Sequence fidelity of chimeric genes --- p.55Chapter 3.7 --- Expression of transgenes in rice --- p.55Chapter 3.7.1 --- Plant materials --- p.55Chapter 3.7.2 --- Agrobacterium transformation --- p.55Chapter 3.7.3 --- Callus induction --- p.56Chapter 3.7.4 --- Agrobacterium culture and rice transformation --- p.56Chapter 3.7.5 --- Selection and regeneration of rice callus --- p.56Chapter 3.7.6 --- Isolation of genomic DNA --- p.58Chapter 3.7.7 --- Southern blot analysis --- p.58Chapter 3.7.8 --- Extraction of leaf total RNA --- p.59Chapter 3.7.9 --- Extraction of seed total RNA --- p.59Chapter 3.7.10 --- Northern blot analysis --- p.60Chapter 3.7.11 --- Protein extraction and Tricine SDS-PAGE --- p.60Chapter 3.7.12 --- Western blot analysis --- p.61Chapter 3.8 --- Cytopathic effect (CPE) reduction assay --- p.61Chapter 3.8.1 --- Protein extraction --- p.61Chapter 3.8.2 --- CPE reduction assay --- p.62Chapter 3.9 --- Confocal immunofluorescence --- p.63Chapter 3.9.1 --- Preparation of sections --- p.63Chapter 3.9.2 --- Labelling of fluorescence probes --- p.63Chapter 3.9.3 --- Image collection --- p.64Chapter Chapter 4 --- Results --- p.65Chapter 4.1 --- Cloning of POL cDNA from Yu Chu --- p.65Chapter 4.1.1 --- RNA extraction and partial POL cDNA amplification --- p.65Chapter 4.1.2 --- 5'RACE and 3'RACE --- p.67Chapter 4.1.3 --- Sequencing of POL cDNA --- p.68Chapter 4.1.4 --- Sequences comparison of POL and Liliaceae lectins --- p.75Chapter 4.2 --- Occurence of POL protein in Yu Chu plant --- p.77Chapter 4.3 --- Constitutional expression of POL in rice --- p.79Chapter 4.3.1 --- Construction of Cauliflower mosaic virus 35S promoter constructs --- p.80Chapter 4.3.2 --- Southern blot analysis --- p.82Chapter 4.3.3 --- Northern blot analysis --- p.84Chapter 4.3.4 --- Western blot analysis --- p.85Chapter 4.3.5 --- Western blot analysis of 35S/POL T1 plant --- p.87Chapter 4.4 --- Seed-specific expression of POL in rice --- p.88Chapter 4.4.1 --- Construction of the glutelin-1 promoter constructs --- p.89Chapter 4.4.2 --- Southern blot analysis --- p.92Chapter 4.4.3 --- Northern blot analysis --- p.96Chapter 4.4.4 --- Western blot analysis --- p.101Chapter 4.4.5 --- Western blot analysis of POL-BP-8O and POL-α-TIP T1 transgenic plants --- p.117Chapter 4.5 --- Cytopathic effect (CPE) reduction assay --- p.122Chapter 4.6 --- Confocal immunofluorescence studies --- p.125Chapter Chapter 5 --- Discussion --- p.134Chapter 5.1 --- Cloning of POL cDNA --- p.134Chapter 5.2 --- Analysis of constitutional expression of POL in rice --- p.136Chapter 5.3 --- Analysis of seed-specific expression of POL in rice --- p.138Chapter 5.4 --- Localization of POL in POL-BP-8O and POL-α-TIP transgenic rice seeds --- p.146Chapter 5.5 --- Cytopathic effect (CPE) reduction assay --- p.148Chapter 5.6 --- Future prospects --- p.151Chapter Chapter 6 --- Conclusion --- p.15