University of Bath PHD Isolation of novel expressed DNA sequences from cassava (Manihot esculenta, Crantz) Marello, Karen Louise Award date: 2000 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 04. Oct. 2021 ISOLATION OF NOVEL EXPRESSED DNA SEQUENCES FROM CASSAVA (MANIHOT ESCULENTA, CRANTZ) submitted by Karen Louise Marello for the degree of PhD of the University of Bath 2000 COPYRIGHT Attention is drawn to the fact that copyright of this thesis rests with its author. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without the prior written consent of the author. This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purposes of consultation. 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Box 1346 Ann Arbor, Ml 48106-1346 UNIVERSITY OF BATH LIBRARY & - 3 OCT 2001 -Ti-fco CONTENTS Page Abstract .............................................................................................................. 1 Acknowledgements ............................................................................................ 2 List of abbreviations ................................................. 3 CHAPTER 1 GENERAL INTRODUCTION ........................................................ 5 1.1 Food security and cassava ................................................................... 5 1.2 Biotechnological applications and the launch of the CBN .................... 8 1.2.1 The storage root - a source of starch ........................................... 9 1.2.2 Cyanogenesis ............................................................................ 13 1.2.3 Post-harvest deterioration .......................................................... 17 1.2.4 Pests and diseases ................... 20 1.2.5 Availability of improved planting material .................................... 23 1.2.6 Conservation of cassava diversity ............................................... 24 1.2.7 Genetic modification of cassava ................................................. 26 1.3 Overview of this project ...................................................................... 27 1.3.1 Storage proteins found in other roots and tubers ........................ 28 1.3.2 Expression patterns ................................................................... 29 1.3.3 Roles beyond the storage of nitrogen and sulphur ...................... 29 1.3.3.1 Patatin ........................................................................................ 29 1.3.3.2 Sporamin ................................................................................... 30 1.3.3.3 Dioscorin ................................................................................... 30 1.3.4 Cassava storage proteins ........................................................... 31 1.3.5 Investigation of a smalt cassava protein located in the storage root ........................................................................................... 31 i 1.3.6 Alternative strategy for the identification of clones expressed in the storage root .......................................................................... 31 CHAPTER 2 MATERIALS AND METHODS .................................................. 33 2.1 Cassava growth conditions ................................................................. 33 2.1.1 Growth period ............................................................................. 33 2.1.2 Nutrient regimes ......................................................................... 33 2.1.3 Pesticides ................................................................................... 34 2.1.4 Harvest of storage root and leaf tissue ........................................ 35 2.2 cDNA library construction .................................................................... 35 2.2.1 mRNA preparation ................................................................ :... 35 2.2.1.1 Pre-treatment of equipment ........................................................ 35 2.2.1.2 Total RNA extraction .................................................................. 36 2.2.1.3 RNA gel electrophoresis ............................................................. 37 2.2.1.4 Purification of total RNA preparation ........................................... 38 2.2.1.5 Isolation of poly (A+) RNA ........................................................... 38 2.2.1.6 RNA quantification ...................................................................... 40 2.2.2 cDNA synthesis .......................................................................... 40 2.2.3 Addition of adaptors to cDNA .................................................... 41 2.2.4 Kinasing reaction ....................................................................... 42 2.2.5 Ligation into Xgt11 ....................................................................... 42 2.2.6 Packaging of the bacteriophage .................................................. 43 2.3 cDNA library screening ........................................................................ 43 2.3.1 Plating cells preparation ............................................................. 43 2.3.2 Titration ...................................................................................... 44 2.3.3 Antibody screening .................................................................... 45 2.3.3.1 Antibody characterization .......................................................... 45 ii 2.3.3.1.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) ............................................................................ 45 2.3.3.1.2 Coomassie staining of SDS-PAGE gels ..................................... 46 2.3.3.1.3 Ponceau S staining of SDS-PAGE gels ...................................... 46 2.3.3.1.4 Electro-transfer of proteins to nitrocellulose membrane .............. 46 2.3.3.1.5 Membrane blocking .................................................................... 46 2.3.3.1.6 Primary antibody binding ............................................................ 47 2.3.3.1.7 Secondary antibody detection .................................................... 47 2.3.3.2 Plate lifts of cDNA expression libraries ....................................... 48 2.3.3.3 Antibody screening .................................................................... 48 2.3.3.4 Secondary antibody detection .................................................... 48 2.3.3.4.1 DAB ........................................................................................... 48 2.3.3.4.2 Electro-Chemiluminescence (ECL) ............................................ 48 2.3.3.5 PCR detection of recombinant bacteriophage ............................ 49 2.3.3.5.1 Miniprep of phage DNA from agar plaques ................................. 50 2.3.3.5.2 PCR of XMOSE/oxcDNA library clones ...................................... 51 2.3.3.5.3 PCR of Xgt11 cDNA library clones .............................................. 51 2.4 cDNA clone analysis .......................................................................... 52 2.4.1 Agarose gel electrophoresis ....................................................... 52 2.4.1.1 TBE gel electrophoresis ............................................................. 52 2.4.1.2 TAE gel electrophoresis ............................................................. 53 2.4.2 DNA gel band purification ........................................................... 53 2.4.2.1 Electroelution ............................................................................. 53 2.4.2.2 Geneclean III DNA purification ................................................... 54 2.4.2.3 Sephaglas DNA purification ........................................................ 55 2.4.3 Sub-cloning