Studies on Regulation of Lignin Biosynthesis gene(s) in Leucaena leucocephala A THESIS SUBMITTED TO THE UNIVERSITY OF PUNE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY BY SUMITA OMER UNDER THE GUIDANCE OF Dr. B. M. KHAN DIVISION OF PLANT TISSUE CULTURE NATIONAL CHEMICAL LABORATORY PUNE – 411 008 INDIA November, 2011 Dedicated to my loving family TABLE OF CONTENTS_________________________________________________ Content Page Acknowledgements . i-ii Declaration . iii Certificate . iv Abstract . v-vii Abbreviations . viii-x CHAPTER1: Introduction . 1-33 1.1 Leucaena leucocephala . 2 1.2 Lignin biosynthesis in nature . 7 1.3 Regulation of lignin biosynthesis . 15 1.4 MYB transcription factors in plants . …22 1.5 Rationale of the thesis . .. 33 CHAPTER2: Materials and Methods . 34-79 2.1 Plant Material . .. 34 2.2 Bacterial strains . 35 2.3 Plasmid vectors used . 35 2.4 Glasswares . 36 2.5 Plastic ware . .36 2.6 Chemicals . .36 2.7 Equipments used for the study . 37 2.8 Buffers and Solutions . 38 2.8.1 Buffers and solutions for DNA electrophoresis. .38 2.8.2 Buffers and solutions for gDNA isolation, Southern blotting and hybridisation. 38 2.8.3 Buffers and solutions for Plasmid isolation (Alkaline lysis method) . 39 2.8.4 Buffers and solutions for Protein Gel Electrophoresis (PAGE). 39 2.8.5 Buffers for protein extraction and purification under denaturing conditions. .41 2.8.6 Buffers for protein extraction and purification under native conditions. .41 2.8.7 Different media and buffers used for bacterial studies. 42 2.8.8 Buffers and solutions for ELISA/Immuno-cytolocalisation/GUS Assay. 42 2.8.9 Components of Murashige and Skoog media/ hormone and antibiotic stock solutions. 43 2.8.10 Different media used for Nicotiana tabacum tissue culture. 44 2.8.11 DNA and protein markers used. 45 2.9 Methods . 46 2.9.1 Bacterial culture conditions. .46 2.9.2 Bacterial transformation. 46 2.9.2.1 Preparation of competent cells using TB buffer . 46 2.9.2.2 Preparation of competent cells using CaCl 2 . 46 2.9.2.3 E.coli transformation. 46 2.9.2.4 Agrobacterium tumefaciens transformation and selection. 47 2.9.2.5 Colony screening by PCR . 47 2.9.3 Preservation of bacteria. 48 2.9.4 Isolation of nucleic acids and Polymerase Chain Reaction (PCR) . 48 2.9.4.1 Isolation of plasmid DNA from E.coli cells. 48 2.9.4.2 Isolation of plant genomic DNA. 49 2.9.4.3 Restriction digestion of DNA. 50 2.9.4.4 Extraction and purification of DNA from agarose gels. 50 2.9.4.5 RNA extraction. 51 2.9.4.6 mRNA purification. 51 2.9.4.7 Spectrophotometric determination of nucleic acid concentration. 52 2.9.4.8 First strand cDNA synthesis by Reverse Transcription . 53 2.9.4.9 Polymerase Chain Reaction (PCR) . 54 2.9.4.10 Rapid Amplification of cDNA ends (RACE) . 55 2.9.5 Isolation of nuclear proteins. 59 2.9.6 Electrophoretic Mobility Shift Assay (EMSA) . 61 2.9.7 Detection of biotin-labeled DNA by Chemiluminescence. 63 2.9.8 Quantitative Real Time PCR (QRT PCR) . .64 2.9.8.1 Pre-protocol considerations. 65 2.9.8.2 Preparing the QRT-PCR reactions. 67 2.9.9 Nucleic acids hybridization. 67 2.9.9.1 Southern hybridization. 68 2.9.9.2 Random primer labeling. 69 2.9.10 Hybridization. 70 2.9.11 Expression and purification of recombinant protein. 70 2.9.11.1 Screening for over-expressing recombinant Ll MYB-SSM protein . 70 2.9.11.2 Protein isolation from inclusion body. 71 2.9.11.3 Affinity Purification of Recombinant Protein Using Ni+ NTA Beads. 71 2.9.11.4 Affinity purification of pET41a (+) cloned recombinant protein using GST•Bind TM resins. 72 2.9.11.5 Polyacrylamide gel electrophoresis (PAGE) . 73 2.9.11.6 Preparation of the separating gel. 73 2.9.11.7 Preparation of the stacking gel . 73 2.9.11.8 Preparation of the sample. 73 2.9.11.9 Loading and running the polyacrylamide gel. .. 74 2.9.11.10 Silver staining of the gel Solutions . 74 2.9.12 Histology and Immuno-cytolocalization . 74 2.9.13 Histochemical Staining . 75 2.9.14 ELISA (Enzyme-Linked Immunosorbent Assay) . ... 75 2.9.14.1 Determination of titre of antibodies . 75 2.9.14.2 ELISA of Ll MYB_SSM protein in different tissues of Leucaena leucocephala . 76 2.9.15 Agrobacterium mediated tobacco transformation . 77 2.9.16 GUS histochemical assay . 78 2.9.17 BRADFORD PROTEIN ASSAY . 78 CHAPTER 3: Cloning and characterisation of promoter sequences for lignin biosynthetic pathway genes (C4H, CCoAOMT, CCR AND CAD) FROM Leucaena leucocephala . 80-107 3.1 Introduction . 80 3.2 Materials and methods . 81 3.3 Results and Discussion . ..