UNIVERSITÀ DEGLI STUDI DI MILANO PhD program in Biochemical Sciences XXIX cycle DeFENS - Department of Food, Environmental and Nutritional Sciences BIOCHEMICAL FUNCTIONAL CHARACTERIZATION AND MOLECULAR BIOLOGY OF PLANT INHIBITOR PROTEINS ACTING AGAINST GLYCOSIDE HYDROLASE Elisabetta GALANTI Tutor: Prof. Alessio SCARAFONI Director of the PhD program: Prof. Sandro SONNINO A.A. 2015-2016 2 C’accht Crist n’de lupin? 3 Sommario ABBREVIATIONS ................................................................................................................................................ 1 INTRODUCTION ................................................................................................................................................. 3 1. Plant cell wall: ........................................................................................................................................... 3 1.1 Structure ............................................................................................................................................ 3 1.1.1 Cellulose .................................................................................................................................... 3 1.1.2 Pectins ....................................................................................................................................... 4 1.1.3 Hemicellulose ............................................................................................................................ 4 1.2 Biosynthesis ...................................................................................................................................... 8 1.2.1 Cellulose .................................................................................................................................... 8 1.2.2 Pectin ......................................................................................................................................... 8 1.2.3 Hemicellulose ............................................................................................................................ 8 1.3 Functions ......................................................................................................................................... 10 2. Enzyme .................................................................................................................................................... 12 2.1 Cell wall modified enzyme .............................................................................................................. 12 2.1.1 GH ............................................................................................................................................ 12 2.1.2 XHT .......................................................................................................................................... 14 2.1.3 Expansins ................................................................................................................................. 15 2.1.4 GT ............................................................................................................................................. 16 2.2 Cell wall degrading enzymes (CWDEs) ........................................................................................... 16 2.2.1 Clan A ....................................................................................................................................... 17 2.2.2 Clan C ....................................................................................................................................... 18 2.2.3 Not classified clan ................................................................................................................... 20 2.2.4 Elicitor ...................................................................................................................................... 20 3. Inhibitor ................................................................................................................................................... 22 3.1 Dicots ............................................................................................................................................... 22 3.2 Cereals ............................................................................................................................................. 25 3.2.1 TAXIs-like ................................................................................................................................. 26 3.2.2 XIPs-like ................................................................................................................................... 27 3.2.3 TLXIs-like ................................................................................................................................. 28 3.2.4 Legumes................................................................................................................................... 29 AIM OF THE WORK .......................................................................................................................................... 31 MATERIALS AND METHODS............................................................................................................................ 32 1. Bioinformatics analysis ........................................................................................................................... 32 1.1 Plant material .................................................................................................................................. 32 1.1.1 Conditions of Arabidopsis thaliana growth in soil ................................................................ 32 4 1.1.2 Conditions of Arabidopsis thaliana growth in solid media (Petri dishes) ............................. 32 1.2 Elicitation of defence responses during seed germination ........................................................... 33 1.2.1 Chitosan preparation .............................................................................................................. 33 1.2.2 GH2 .......................................................................................................................................... 33 1.2.3 GH5 .......................................................................................................................................... 33 1.2.4 Paenibacillus polymyxa .......................................................................................................... 33 1.3 Genomic Analysis ............................................................................................................................ 34 1.3.1 Extraction and purification of genomic DNA from Arabidopsis thaliana ................................. 34 1.3.2 Extraction and purification of total RNA from Arabidopsis thaliana .................................... 34 1.3.3 DNA electrophoresis on agarose gels ..................................................................................... 34 1.3.4 Polymerase Chain Reaction (PCR) for genotyping ................................................................. 34 1.3.5 Synthesis of cDNA by reverse transcription (RT) ................................................................... 35 1.3.6 Quantitative Real-Time Polymerase Chain Reaction (PCR) for expression analysis ............ 35 1.4 Proteomic Analysis ......................................................................................................................... 36 1.4.1 Total protein extraction (TPE) .................................................................................................... 36 1.4.2 Albumin and globulin purification.......................................................................................... 36 1.4.3 γ-Conglutin purification .......................................................................................................... 37 1.4.4 Proteins exudate purification ................................................................................................. 37 1.4.5 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ............................................................ 37 1.4.6 Western blot Analyses ............................................................................................................ 38 1.5 Enzymatic assay .............................................................................................................................. 38 1.5.1 GH11 ........................................................................................................................................ 38 1.5.2 GH5 .......................................................................................................................................... 38 1.5.3 GH2 .......................................................................................................................................... 39 1.5.4 p-hydroxy-benzoic acid hydrazide Method ........................................................................... 39 1.5.5 Inhibitory assays ..................................................................................................................... 39 1.5.6 Size exclusion chromatography (SEC)..................................................................................... 40 RESULTS AND DISCUSSIONS ........................................................................................................................... 41 1.1 Bioinformatics Analysis .................................................................................................................