ESV1) Protein and Its Homologue Like- Early Starvation 1 (LESV) During Starch Degradation

ESV1) Protein and Its Homologue Like- Early Starvation 1 (LESV) During Starch Degradation

Biochemical studies to determine the role of Early Starvation 1 (ESV1) protein and its homologue Like- Early Starvation 1 (LESV) during starch degradation Dissertation zur Erlangung des akademischen Grades "doctor rerum naturalium" (Dr. rer. nat.) in der Wissenschaftsdisziplin Biochemistry eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät Institut für Biology/Biochemistry der Universität Potsdam von Shadha Abduljaleel AL-Rawi Potsdam. 2020 Main supervisor: Prof Dr. habil. Joerg Fettke Supervisors: Prof. Dr. Alisdair Fernie Reviewers: Prof. Dr. Christophe D’Hulst Prof. Dr. Oluwatoyin A. Odeku Published online on the Publication Server of the University of Potsdam: https://doi.org/10.25932/publishup- 48395 https://nbn-resolving.org/urn:nbn:de:kobv:517-opus4- 483956 Table of contents Table of contents Table of contents ................................................................................................................ 1 Abstract ............................................................................................................................... 5 Zusammenfassung ............................................................................................................. 7 Acknowledgement .............................................................................................................. 9 List of Figures .................................................................................................................. 11 List of Tables .................................................................................................................... 13 Abbreviation ..................................................................................................................... 14 1. Introduction .................................................................................................................. 18 1.1 Carbohydrates ........................................................................................................... 18 1.2 Classification of carbohydrates .................................................................................. 18 1.2.1 Monosaccharides ........................................................................................................................ 18 1.2.2 Disaccharides .............................................................................................................................. 19 1.2.3 Oligosaccharides ......................................................................................................................... 20 1.2.4 Polysaccharides ........................................................................................................................... 21 1.3 Starch and glycogen are the two important storage biopolymers in nature ............... 22 1.3.1 Glycogen ..................................................................................................................................... 22 1.3.2 Starch .......................................................................................................................................... 23 1.4 Starch synthesis ......................................................................................................... 25 1.4.1 Adenosine 5, diphosphate glucose pyrophosphorylase (AGPase) E.C. 2.7.7.27 ........................ 25 1.4.2 Starch Synthases (SSs) E.C. 2.4.1.21 .......................................................................................... 26 1.4.3 Starch branching enzymes (SBEs) E.C. 2.4.1.18 ....................................................................... 27 1.4.4 Starch debranching enzymes DBEs E.C. 3.2.1.41 ..................................................................... 29 1.5 Disproportionating enzyme D-enzyme E.C. 2.4.1.25 .................................................. 29 1.6 Alpha glucan phosphorylase ...................................................................................... 31 1.7 Starch degradation .................................................................................................... 32 1.8 Metabolism of starch ................................................................................................. 41 1.9 The biological function for complex formation of starch metabolic enzymes ............. 42 1.10 Putative protein-protein interaction within starch granules..................................... 44 1.11 Novel proteins influence starch metabolism ............................................................. 45 1.11.1 Regulatory proteins in starch synthesis ..................................................................................... 45 1.11.2 Regulatory proteins in starch degradation................................................................................. 47 1.12 The aims of the study ............................................................................................... 51 2. Materials and Methods ................................................................................................ 53 2.1 Materials ................................................................................................................... 53 1 Table of contents 2.1.1 Plant material and growth conditions.......................................................................................... 53 2.1.2 Starch isolation ........................................................................................................................... 53 2.1.3 Chemicals ................................................................................................................................... 54 2.1.4 Enzymes ...................................................................................................................................... 54 2.2 Methods ..................................................................................................................... 54 2.2.1 Preparation of E.coli LB21 competent cells ............................................................................... 54 2.2.2 Preparation of E.coli DH5 ɑ competent cells .............................................................................. 54 2.2.3 Preparation of agarose gel electrophoresis .................................................................................. 55 2.2.4 Preparation of SDS-PAGE .......................................................................................................... 55 2.2.5 Western blot ................................................................................................................................ 56 2.2.6 Detection of Like-Early Starvation 1 protein LESV in Arabidopsis thaliana ............................ 56 2.2.7 Sequence identification of Like-Early Starvation 1 protein LESV in Arabidopsis thaliana genome................................................................................................................................................. 57 2.2.8 Cloning of Arabidopsis LESV protein ........................................................................................ 58 2.2.9 Agarose-gel electrophoresis ........................................................................................................ 60 2.2.10 DNA-Extraction from agarose gel (GFX PCR DNA and GEL Band Purification Kit) ............ 60 2.2.11 Ligation of DNA fragments ...................................................................................................... 60 2.2.12 Transformation of plasmid DNA in competent E.coli cells ...................................................... 61 2.2.13 Colony PCR .............................................................................................................................. 61 2.2.14 Plasmid preparation (Miniprep) ................................................................................................ 62 2.2.15 Restriction with endonucleases ................................................................................................. 62 2.2.16 Quantification of DNA ............................................................................................................. 63 2.2.17 Sequencing ................................................................................................................................ 63 2.2.18 Subcloning of LESV sequence into pET23B expression vector ............................................... 63 2.2.18.1 Restriction digestion for cutting the target sequence and vector ....................................... 63 2.2.18.2 Ligation of LESV sequence into pET23b expression vector ............................................. 64 2.2.18.3 Minipreparation ................................................................................................................. 65 2.2.19 Cloning of LESV without transit peptide (tLESV) ................................................................... 65 2.2.20 Expression and small scale purification of LESV and tLESV in E.coli BL21 cells ................. 66 2.2.21 Preparation of glycerol stock for E.coli BL21 cells containing the plasmid with the protein of interest ................................................................................................................................................. 67 2.2.22 Expression and purification of recombinant LESV and tLESV (large scale) ........................... 67 2.2.23 Measuring proteins concentration ............................................................................................. 68 2.2.24 Protein

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