Molecular Characterisation of Insect Amylases and Plant Amylase Inhibitors
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Molecular Characterisation of Insect Amylases and Plant Amylase Inhibitors Thesis Submitted to AcSIR For the Award of the Degree of DOCTOR OF PHILOSOPHY In BIOLOGICAL SCIENCES By Amey Jayant Bhide 10BB11J26109 Under the guidance of Dr. Ashok P. Giri (Research Supervisor) Dr. Vidya S. Gupta (Research Co-supervisor) BIOCHEMICAL SCIENCES DIVISION CSIR-NATIONAL CHEMICAL LABORATORY PUNE-411008, INDIA August 2016 Dedicated to…………….. Aai, Baba & Family PhD Thesis Table of Contents Acknowledgement i List of tables iv List of figures v Abbreviations viii Abstract x Chapter 1: Introduction 1.1 Influence of insect pests on agriculture 1 1.2 Digestive physiology of insects: Complexity in expression and 5 importance in gaining nutritional requirements 1.3 Targeting insect pests digestive enzymes with plant proteinaceous 10 inhibitors 1.4 Molecular insights into mechanism of action of inhibitors against target 13 enzymes 1.5 Molecular insights into mechanism of induced resistance of insect against 22 plant proteinaceous inhibitors 1.6 Regulation of endogenous and transgenic proteinaceous inhibitors in 23 plants 1.7 Advances in utilization of plant proteinaceous inhibitors against insect 24 digestive enzymes Chapter 2: Gene cloning and expression of Helicoverpa armigera amylases 2.1 Introduction 27 2.2 Materials and Methods 2.2.1 Insect culture 28 2.2.2 Feeding assays 28 2.2.3 RNA isolation, cDNA synthesis and real time PCR 29 2.2.4 Protein sequence analyses 30 2.2.5 Estimation of starch and sugars 30 2.2.6 α-Amylase activity in oral secretions, haemolymph and 30 faecal matter 2.2.7 Statistical analysis 31 2.3 Results 2.3.1 Sequence analysis of HaAmy1 and HaAmy2 31 2.3.2 Differential expression of HaAmy1 and HaAmy2 genes 31 during insect development Amey J Bhide PhD Thesis 2.3.3 Tissue specific expression of HaAmy1 and HaAmy2 33 genes 2.3.4 HaAmy1 and HaAmy2 gene expression is influenced by 34 reducing sugar, sucrose and starch availability 2.3.5 Expression of HaAmy1 and HaAmy2 genes during larval 37 development 2.3.6 α-Amylase levels in the gut, oral secretion, haemolymph 39 and faecal matter 2.4 Discussion 39 2.5 Summary 44 Chapter 3: Biochemical, structural and functional diversity among Helicoverpa armigera amylases 3.1 Introduction 45 3.2 Materials and Methods 3.2.1 Insect culture 46 3.2.2 RNA extraction and cDNA synthesis 47 3.2.3 Cloning of HaAmy1 and HaAmy2, expression and 47 purification 3.2.4 Homology modeling of HaAmy1 and HaAmy2 47 3.2.5 α-Amylase-inhibitor docking and molecular dynamics 48 simulations 3.2.6 In-gel α-amylase activity and molecular weight 48 determination 3.2.7 Determination of α-amylase and α-amylase inhibitory 49 activity 3.2.8 End product analysis of starch hydrolysis 49 3.2.9 Effect of pH and temperature on the activity of enzyme 50 3.2.10 Determination of kinetic parameters 50 3.2.11 Circular Dichroism spectroscopy 50 3.2.12 FPLC analysis of complexes between HaAmy1 and 51 HaAmy2 with wheat α-amylase inhibitor 3.3 Results 3.3.1 Sequence analysis and homology modeling of HaAmy1 51 and HaAmy2 3.3.2 Recombinant protein expression, purification and 55 activity of HaAmy1 and HaAmy2 3.3.3 Biochemical properties of HaAmy1 and HaAmy2 vary 57 with respect to substrates 3.3.4 Higher thermal conditions have minor effect on 58 biochemistry and conformational stability of HaAmy1 and HaAmy2 3.3.5 HaAmy1 and HaAmy2 were stable under a wide range 62 of pH Amey J Bhide PhD Thesis 3.3.6 Proteinaceous and non-proteinaceous α-amylase 63 inhibitor had differential activity towards recombinant HaAmy1 and HaAmy2 3.3.7 Inhibitor docking 64 3.4 Discussion 67 3.5 Summary 68 Chapter 4: Characterization of two Coleopteran α-amylase and molecular insights into their differential inhibition by synthetic α-amylase inhibitor, acarbose 4.1 Introduction 69 4.2 Materials and Methods 4.2.1 Phylogenetic analysis of Coleopteran α-amylases 70 4.2.2 Insect rearing 70 4.2.3 RNA extraction and cDNA synthesis 71 4.2.4 Cloning, expression and purification of CcAmy and 71 TcAmy 4.2.5 Homology modeling and docking studies of CcAmy and 72 TcAmy 4.2.6 In-gel α-amylase activity and molecular weight 73 determination 4.2.7 Determination of α-amylase activity 74 4.2.8 Effect of pH and temperature on the activity of enzyme 74 4.2.9 Analysis of end products of starch hydrolysis 74 4.2.10 Determination of kinetic parameters and α-amylase 75 inhibitory activity 4.3 Results 4.3.1 Phylogenetic analysis of Coleopteran α-amylases 75 4.3.2 Homology modeling and docking analysis of CcAmy 76 and TcAmy 4.3.3 Amylolytic products and molecular weight determination 77 of recombinant CcAmy and TcAmy 4.3.4 Kinetic parameters for CcAmy and TcAmy 79 4.3.5 Biochemical properties of CcAmy and TcAmy 81 4.3.6 Interactions of proteinaceous and non-proteinaceous 83 inhibitors with CcAmy and TcAmy 4.4 Discussion 89 4.5 Summary 91 Amey J Bhide PhD Thesis Chapter 5: Genomic and functional characterization of coleopteran insect specific α- amylases inhibitor gene from Amaranthus species 5.1 Introduction 93 5.2 Materials and Methods 5.2.1 Sequence and phylogenetic analysis of α-amylase 95 inhibitors 5.2.2 RNA isolation, cDNA synthesis and real time PCR 95 5.2.3 Sequence analysis of 3’UTR region 96 5.2.4 Cloning, expression and purification of AhAI 96 5.2.5 Localization of GFP fused with AhAI signal peptide 97 5.2.6 Determination of accurate molecular weight and α- 98 amylase inhibitory activity 5.2.7 Insect rearing and feeding assay 98 5.2.8 Circular Dichroism spectroscopy and homology 99 modelling of AhAI 5.2.9 Docking studies of AhAI with CcAmy and TcAmy 100 5.3 Results 5.3.1 Gene structure and comparison of AhAI with other α- 101 amylase inhibitors 5.3.2 AhAI orthologs from Amaranthaceae members exhibit 102 identical open reading frame while variation in 3’UTR sequence 5.3.3 AhAI transcript abundance varies across the plant parts 103 of Amaranthaceae members 5.3.4 AhAI signal peptide mediates extracellular transport 106 5.3.5 AhAI is a potent inhibitor of coleopteran α-amylases 107 5.3.6 Differential interaction might be responsible for 109 concentration dependent inhibition of coleopteran α- amylases 5.4 Discussion 111 5.5 Summary 115 Chapter 6: Summary & Future prospective 6. Summary and Future prospective 117 Bibliography 121 List of Publications 156 Curriculum Vitae Amey J Bhide PhD Thesis Acknowledgement This thesis is the outcome of several people’s contribution, without their support this wonderful journey, which is a blend of multitude of things, learning, patience, help, encouragement, aspiration, ecstasy and melancholy would have not been possible. Hence, it becomes my innermost will to acknowledge all those who have extended their support to me scientifically, morally or otherwise to make my way smooth. First and foremost, I owe to my parents and my family members who have walked through myriads of rough patches to pave my way and have been always there for everything in my ups and downs. I am extremely grateful to Dr. Ashok P. Giri, my research guide, for providing a great support and having faith in me. This work would not have been possible without his guidance, critical inputs and freedom for executing my ideas as well. He has always supported me in every sense throughout these years. He has followed my progress from the first day and helped in a great deal in improving my speaking skill. I also appreciate his meticulous attention to the minute details. I appreciate all his effort, ideas and time to make this happen. I also appreciate his friendly and helpful nature in the lab. I am also grateful to Dr. Hemlata Kotkar for her inevitable support and encouragement which have accelerated pace of my work and moved to next step. I also acknowledge my Co-guide Dr. (Mrs.) Vidya S. Gupta for kindly replying my queries and giving suggestions out of her busy schedule for my work. She has been associated with my research work from the beginning and has provided critical inputs and suggestions. I am also thankful to Dr. Narendra Y. Kadoo for his critical suggestions and proof reading of my NCL-401 and NCL-402 project works. I am thankful to my DAC chairman Dr. Mugdha Gadgil and DAC members Dr. Avalokiteshwar Sen, Dr. Narendra Y. Kadoo and Prof. Kisan M. Kodam (SPPU) for their constant support, encouragement and suggestions during DAC meetings. I express my gratitude towards Dr. Sureshkumar Ramasamy for helping me in computational analysis and also for his useful suggestions on protein purification. I am also thankful to Prof. V. L. Maheshwari and Dr. Pankaj K. Pawar for their valuable suggestions on biochemical assays and insect feeding experiments. I am grateful to Dr. R. V. Gadre for allowing me to use HPLC facility and also teaching me basics of fermentation and good laboratory practices during my tenure as a project fellow in his lab. I am thankful to Dr. Mahesh Kulkarni for Amey J. Bhide i PhD Thesis giving his valuable advice on proteomics work and also to Dr. Suresh Bhat for allowing me to use confocal microscope. I express special thanks to Dr. Gayathri from IISER-Pune for her help in crystal data collection. I am grateful to Dr. Yogesh Shouche for allowing me to work in his lab as a project trainee during my M.