by Chow Fung-cheung, Judy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 106-117). / ACKNOWLEDGMENT --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / LIST OF ABBREVIATIONS --- p.ix / LIST OF FIGURES AND TABLES --- p.xii / Chapter SECTION I- --- ISOLATION OF ACTIN GENE FRAGMENTS FROM CHLORELLA VULGARIS / Chapter CHAPTER 1: --- INTRODUCTION / Chapter 1.1 --- Functions of Actin --- p.1 / Chapter 1.1.1 --- Functions of Actin in Animals --- p.1 / Chapter 1.1.2 --- Functions of Actin in Plants --- p.2 / Chapter 1.1.2.1 --- In Lower Plants --- p.2 / Chapter 1.1.2.2 --- In Higher Plants --- p.2 / Chapter 1.2 --- Molecular Studies of Actin Gene Families in Plants --- p.4 / Chapter 1.2.1 --- Multigene Family --- p.4 / Chapter 1.2.2 --- Homologies Across Kingdom --- p.4 / Chapter 1.2.3 --- Homologies Within Kingdom --- p.5 / Chapter 1.2.4 --- Position of Intron --- p.5 / Chapter 1.2.5 --- Differential Expression of Actin Genes --- p.7 / Chapter 1.3 --- Objectives of Present Studies --- p.7 / Chapter CHAPTER 2: --- GENERAL TECHNIQUES / Chapter 2.1 --- Growth of Algal Strain --- p.9 / Chapter 2.2 --- Growth of Bacterial Strains --- p.10 / Chapter 2.3 --- Agarose Gel Electrophoresis --- p.10 / Chapter 2.4 --- Restriction Enzyme Digestion --- p.10 / Chapter 2.5 --- Recovery of DNA Fragments from Agarose Gel --- p.11 / Chapter 2.5.1 --- Glass Powder Elution of DNA --- p.11 / Chapter 2.5.2 --- Sephaglas´ёØ BandPrep Kit --- p.11 / Chapter 2.6 --- Large Scale Preparation of Plasmid by Using Magic´ёØ Maxipreps DNA Purification System --- p.12 / Chapter 2.7 --- Ligation --- p.13 / Chapter 2.8 --- Preparation of Competent Cells --- p.13 / Chapter 2.9 --- Transformation of Competent Cells --- p.14 / Chapter 2.10 --- Screening of Recombinant Plasmids --- p.14 / Chapter 2.11 --- Spun Column Techniques --- p.15 / Chapter CHAPTER 3: --- PCR-CLONING OF ACTIN GENE FRAGMENTS FROM CHLORELLA VULGARIS / Chapter 3.1 --- Introduction --- p.16 / Chapter 3.2 --- Materials and Methods --- p.16 / Chapter 3.2.1 --- Preparation of Genomic DNA from C. vulgaris --- p.16 / Chapter 3.2.2 --- Amplification of Actin Genomic Fragments by PCR --- p.17 / Chapter 3.2.3 --- Cloning of PCR Products --- p.17 / Chapter 3.2.4 --- Southern Blotting --- p.18 / Chapter 3.2.5 --- Radiolabeling of DNA Probe --- p.19 / Chapter 3.2.6 --- Prehybridization and Hybridization --- p.19 / Chapter 3.2.7 --- Sequencing Strategies --- p.20 / Chapter 3.2.7.1 --- Isolation of Template DNA --- p.20 / Chapter 3.2.7.2 --- Template Denaturation and Primer Annealing --- p.21 / Chapter 3.2.7.3 --- Labeling and Termination Reaction --- p.21 / Chapter 3.2.7.4 --- DNA Sequencing Electrophoresis --- p.21 / Chapter 3.3 --- Results and Discussion --- p.22 / Chapter CHAPTER 4: --- CLONING OF ACTIN COMPLEMENTARY DNA FRAGMENT FROM CHLORELLA VULGARIS / Chapter 4.1 --- Introduction --- p.31 / Chapter 4.2 --- Materials and Methods --- p.31 / Chapter 4.2.1 --- Preparation of RNA --- p.31 / Chapter 4.2.2 --- RT-PCR --- p.32 / Chapter 4.2.3 --- Southern Blotting and Hybridization --- p.32 / Chapter 4.2.4 --- Radiolabeling of DNA Probe --- p.32 / Chapter 4.2.5 --- Cloning of RT-PCR Product --- p.33 / Chapter 4.2.6 --- DNA Sequencing --- p.33 / Chapter 4.2.7 --- Sequence Analysis --- p.33 / Chapter 4.3 --- Results and Discussion --- p.34 / Chapter CHAPTER 5: --- SEQUENCE COMPARISON OF ACTIN GENES / Chapter 5.1 --- Introduction --- p.44 / Chapter 5.2 --- Materials and Methods --- p.44 / Chapter 5.3 --- Results and Discussion --- p.44 / Chapter 5.3.1 --- Nucleotide Sequence Analysis --- p.44 / Chapter 5.3.2 --- Analysis of the Predicted Amino Acid Sequence --- p.49 / Chapter 5.3.3 --- Codon Usage --- p.49 / Chapter 5.3.4 --- Intron-Exon Structure in Plant Actin Genes --- p.51 / Chapter 5.3.5 --- General Discussion --- p.53 / Chapter CHAPTER 6: --- ISOLATION OF FURTHER UPSTREAM SEQUENCE FOR ACTIN GENE (CAc18G) FROM CHLORELLA VULGARIS / Chapter 6.1 --- Introduction --- p.54 / Chapter 6.2 --- Materials and Methods --- p.54 / Chapter 6.2.1 --- Genomic Southern Analysis --- p.54 / Chapter 6.2.2 --- Preparation of Actin-Enriched DNA Fraction --- p.55 / Chapter 6.2.3 --- Ligation of Actin-Enriched Fragments with Specific DNA Cassette --- p.55 / Chapter 6.2.4 --- Amplification of Upstream Sequence by Nested PCR --- p.55 / Chapter 6.2.5 --- DNA Sequencing --- p.56 / Chapter 6.3 --- Results and Discussion --- p.57 / Chapter SECTION II - --- CONSTRUCTION OF TRANSGENIC CASSETTES FOR THE PRODUCTION OF BACILLUS TOXIN IN CHLORELLA VULGARIS / Chapter CHAPTER 1： --- INTRODUCTION / Chapter 1.1 --- Characteristics of Algae --- p.64 / Chapter 1.2 --- Biotechnology Potential of Algae --- p.66 / Chapter 1.3 --- Transgenic Algae --- p.68 / Chapter 1.3.1 --- Genes of Selection for Transformant --- p.70 / Chapter 1.3.1.1 --- Homologous Genes --- p.70 / Chapter 1.3.1.2 --- Heterologous Genes --- p.70 / Chapter 1.3.2 --- Transformation Technologies Used in Algae --- p.71 / Chapter 1.3.3 --- Expression of Transgenes in Algae --- p.73 / Chapter 1.4 --- Bacillus Toxin --- p.73 / Chapter 1.4.1 --- Bacillus thuringiensis --- p.73 / Chapter 1.4.2 --- Classification of Bacillus Toxin Genes (Cry Genes) --- p.74 / Chapter 1.4.2.1 --- Type I (CtyI Genes) --- p.74 / Chapter 1.4.2.2 --- Type II (CryII Genes) --- p.75 / Chapter 1.4.2.3 --- Type III (CryIII Genes) --- p.76 / Chapter 1.4.2.4 --- Type IV (CryIV Genes) --- p.76 / Chapter 1.4.3 --- Mode of Action of Insecticidal Effects --- p.77 / Chapter 1.5 --- Insect-Resistance Transgenic Plants --- p.78 / Chapter 1.5.1 --- Transgenic Plants Expressing Crystal Protein Gene --- p.79 / Chapter 1.5.2 --- Problems Encountered --- p.80 / Chapter 1.6 --- Aims of Present Studies --- p.80 / Chapter CHAPTER 2: --- CONSTRUCTION OF TRANSGENIC CASSETTES / Chapter 2.1 --- Introduction --- p.82 / Chapter 2.2 --- Materials and Methods --- p.82 / Chapter 2.2.1 --- Preparation of Plasmids Involved in the Construction of Master Cassette --- p.82 / Chapter 2.2.2 --- Construction of Master Cassette --- p.83 / Chapter 2.2.3 --- Multiple Cloning Site (MCS) of Master Cassette --- p.83 / Chapter 2.2.4 --- Preparation of Plating Cells --- p.85 / Chapter 2.2.5 --- Titering --- p.85 / Chapter 2.2.6 --- Preparation of Plate Lysate --- p.86 / Chapter 2.2.7 --- Amplification of Coding Region of CryIVC Gene --- p.86 / Chapter 2.2.8 --- Cloning of PCR Products --- p.87 / Chapter 2.2.9 --- Construction of Transgenic Cassette --- p.87 / Chapter 2.2.10 --- Confirmation of the Junction Sites --- p.89 / Chapter 2.2.11 --- Testing for the Sensitivity of Algae Towards Kanamycin --- p.90 / Chapter 2.3 --- Results and Discussion --- p.91 / Chapter CHAPTER 3: --- TRANSFORMATION OF ALGAE BY ELECTROPORATION / Chapter 3.1 --- Introduction --- p.97 / Chapter 3.2 --- Materials and Methods --- p.97 / Chapter 3.2.1 --- Harvesting of Algae --- p.97 / Chapter 3.2.2 --- Electroporation at Different Field Strength --- p.98 / Chapter 3.2.3 --- Plating Culture of Algal Cells --- p.98 / Chapter 3.2.4 --- Preparation of Plasmids for Electrop oration --- p.98 / Chapter 3.2.5 --- Transformation of Algae --- p.100 / Chapter 3.2.6 --- Study on the Uptake of DNA after Electrop oration --- p.100 / Chapter 3.2.6.1 --- Genomic DNA Preparation --- p.100 / Chapter 3.2.6.2 --- Analysis of DNA Uptake --- p.101 / Chapter 3.3 --- Results and Discussion --- p.101 / REFERENCES --- p.106 / APPENDIX --- p.118
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_320667 |
| Date | January 1995 |
| Contributors | Chow, Fung-cheung Judy., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Publisher | Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xiii, 120 leaves : ill. (some mounted col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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