Studies on Engineering Entomopathogenic Fungi, Metarhizium Anisopliae and Beauveria Bassiana to Express Heterologous Insect-Specific Toxins
Total Page：16
File Type：pdf, Size：1020Kb
Department of Biosciences and Bioengineering Indian Institute of Technology Guwahati Guwahati-781039 Assam, India Studies on engineering Entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana to express heterologous insect-specific toxins THESIS Submitted for the partial fulfilment of requirement for the award of DOCTOR OF PHILOSOPHY Under the guidance of by Prof. Gurvinder Kaur Saini DHANASINGH M TH-2353_1361060053 TH-2353_1361060053 Dedicated to My Parents, Family, Loved ones and the Almighty TH-2353_1361060053 TH-2353_1361060053 INDIAN INSTITUTE OF TECHNOLOGY GUWAHATI Department of Biosciences and Bioengineering DECLARATION I do hereby declare that the content embodied in this thesis is the result of investigations carried out by me in the Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, India, under the supervision of Prof. Gurvinder Kaur Saini. In keeping with the general practice of reporting scientific observations, due acknowledgements have been made wherever the work of other investigators are referred. Date: Dhanasingh M Place: Roll No:136106005 i | Page TH-2353_1361060053 TH-2353_1361060053 INDIAN INSTITUTE OF TECHNOLOGY GUWAHATI Department of Biosciences and Bioengineering CERTIFICATE It is certified that the work described in this thesis entitled “Studies on engineering entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana to express heterologous insect-specific toxins” by Mr. Dhanasingh M for the award of degree of Doctor of Philosophy is an authentic record of the results obtained from the research work carried out under my supervision at the Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, India and this work has not been submitted elsewhere for a degree. Date: Dr. Gurvinder Kaur Saini Place: Professor (Thesis supervisor) iii | Page TH-2353_1361060053 TH-2353_1361060053 ACKNOWLEDGEMENT The results embodied in thesis are the outcome of cooperation of many helping hands and it could not have seen the light of the day without their voluntary involvement. Here I take the pleasant opportunity to express my gratitude to all of them. First and foremost, with a deepest sense of respect, I would like to express my sincere gratitude to my supervisor, Dr. Gurvinder Kaur Saini, for her valuable suggestions, encouragement and inspiration. I am indebted to her for introducing me into the realm of this interesting work, training me with the techniques, giving me the freedom in designing and conducting the experiments. I earnestly thank her for imbibing scientific temperament and appreciable work ethics in me which enhanced my grasp of knowledge and made my learning pursuit exciting. I would like to acknowledge my sincere gratitude to all my doctoral committee members, Dr. K. Pakshirajan, Dr. V.V.Dasu, Dr. Siddhartha S. Ghosh, and Dr. C.V.Sastri for their valuable suggestions and precious time. I remain thankful to my seniors Dr. Digar singh for the guidance and training. I am immensely thankful to my research group members Balwant, Neha, Ashish, Tusarika, Kawkab, Jena, Arman, and Arupam for their adept hands in fruition of some of my crucial experiments. Special thanks goes to my friends Muthuvel, Krishnakumar, Anil Kumar, Neha Arora, Himanshu and Yoganand for their academic support and encouragement during my entire journey of PhD Life. I am thankful to Dr. Nurul, Mrs. Prarthana, Mr.Yadav, Mr. Dipankar and Mr. Niranjan for their technical help and assistance. I am thankful to the Indian Institute of Technology Guwahati (IITG) for providing me with the state of the art infrastructure for advance level of research. I also thank Central Instrumentation Facility (CIF) at IITG for providing the instrument facilities, National Bureau of Agricultural Insect Resources (NBAIR, Bangalore) for providing the insect cultures, and National center for Cell Sciences v | Page TH-2353_1361060053 (NCCS), Pune, for providing the cell lines for my research work. I wholeheartedly thank Dr. Daniel R. Zeigler, Bacillus Genetic Stock Center (BGSC), USA, for gifting me the Bacillus culture. I am thankful to the Ministry of Human Resource Development (MHRD), New Delhi for the financial support. Finally, my PhD, endeavor could not have been successful without the unfathomable love, support and blessings of My Parents. I owe my achievements to my sisters and brother. I convey my special thanks to my dearest friend Vartika, without whom, I would have not have been able to cope up the stress of my PhD life. Dhanasingh M August, 2019 vi | Page TH-2353_1361060053 CONTENT List of Tables xvii List of Figures xix List of Abbreviations xxv Synopsis xxix Chapter 1: Introduction, Review of Literature and objectives 1.1 General introduction 3 1.2 Food security challenges associated with agricultural pest and 5 diseases 1.3 Global and Indian scenario of insect pest control 6 1.4 Development of pesticide resistance in insect pests 6 1.4.1 Pre-adaptation hypothesis 7 1.4.2 Enhanced detoxification mechanism 7 1.5 Environmental and human health risks associated with insecticides 8 1.5.1 Negative impact on pollinators 9 1.6 Biological pesticides 9 1.6.1 Bacteria as insect biological control agent 10 1.6.1.1 Bacillus thuringiensis 10 1.6.2 Baculovirus as biocontrol agents 11 1.6.3 Fungi as biological control agents 12 1.7 Entomopathogenic fungi Metarhizium anisopliae and Beauveria 12 bassiana 1.7.1 Metarhizium anisopliae 12 1.7.2 Beauveria bassiana 14 1.7.3 Entomopathogenic fungi life cycle 15 1.8 Major drawbacks associated with entomopathogenic fungi 16 1.9 Strategies used to improve the virulence of entomopathogenic fungi 17 1.9.1 Endogenous genes overexpression 17 vii | Page TH-2353_1361060053 Content 1.9.2 Heterologous genes overexpression 18 1.9.2.1 Scorpion toxins overexpression 18 1.9.2.2 Overexpression of other heterologous proteins 18 1.10 Scorpion and spider toxins 19 1.10.1 Voltage-gated channels 19 1.10.2 β-BUTX Lqq1a toxin 20 1.11 Bacillus thuringiensis Cry and Cyt toxins 20 1.11.1 General aspects of Cry1Ac toxin and insect resistance 22 development 1.12 Cytolytic toxin structure overview 23 1.13 General aspects of Lectins 24 1.13.1 GNA Lectin 25 1.14 Lacunae and scope of work 27 1.15 Objectives 28 Chapter 1: Tables and Figures 29-35 Chapter 2: Expression and purification of β-BUTX Lqq1a toxin and its biological activity against insect cells and agricultural insect pests 2.1 Introduction 39-41 2.2 Materials and Methods 42-50 2.2.1 Bacterial strains and reagents 42 2.2.2 β-BUTX Lqq1a gene synthesis 42 2.2.3 Expression cassette construction 42 2.2.4 E. coli chemical competent cell preparation 43 2.2.4.1 E. coli DH5-α transfection 43 2.2.5 Protein β-BUTX Lqq1a expression and purification 44 2.2.5.1 Shuffle T7 express LysY chemical competent cells 44 preparation 2.2.5.2 The (His)6 β-BUTX Lqq1a toxin expression 44 2.2.5.3 The (His)6 β-BUTX Lqq1a toxin affinity purification 45 viii | Page TH-2353_1361060053 Content 2.2.5.4 The (His)6 tag removal and size exclusion 45 chromatography purification 2.2.6 MALDI-TOF molecular mass determination 46 2.2.7 Western blotting analysis 46 2.2.8 Cytotoxicity assay of purified β-BUTX Lqq1a with Sf-21 insect 47 cell lines 2.2.8.1 MTT assay 47 2.2.8.2 Light microscopy analysis 48 2.2.8.3 Fluorescence microscopy analysis 48 2.2.8.4 Ultra-structural analysis using FE-SEM 49 2.2.8.5 Flow cytometry analysis 49 2.2.9 Bioassay of β-BUTX Lqq1a with H. armigera and S. litura 50 2.2.9.1 Maintenance of insect cultures 50 2.2.9.2 Intrahemocoeilic injection bioassay 50 2.2.10 Statistical data analysis 50 2.3 Results 52-58 2.3.1 Cloning in pET28a expression vector 52 2.3.2 Expression and purification using Ni-NTA affinity 52 chromatography 2.3.3 N-Terminal (His)6 tag removal using Thrombin Protease 53 2.3.4 Size exclusion chromatography 53 2.3.5 Mass spectrometry and Western blotting analysis 54 2.3.6 Cytotoxicity analysis of treated Sf-21 insect cell line using MTT 55 assay 2.3.7 Morphological assessment using Light microscopy 55 2.3.8 Fluorescence microscopy analysis 56 2.3.9 Ultra structural analysis using FE-SEM 56 2.3.10 Flow Cytometry analysis 56 2.3.11 Insect bioassay 57 2.3.11.1 Helicoverpa armigera 57 2.3.11.2 Spodoptera litura 57 ix | Page TH-2353_1361060053 Content 2.4 Discussion 59-62 2.5 Conclusion 63 Chapter 2: Tables and Figures 64-75 Chapter 3: Cloning and expression of β-BUTX Lqq1a in Beauveria bassiana and Metarhizium anisopliae and insect bioassay 3.1 Introduction 79-81 3.2 Materials and Methods 82-94 3.2.1 Strains and culture conditions 82 3.2.1.1 Fungal spore collection 82 3.2.2 Gene synthesis 82 3.2.3 Vector construction 83 3.2.3.1 Vector construction for B. bassiana 83 3.2.3.1.1 Overlap extension PCR 83 3.2.3.1.2 E. coli chemical competent cell preparation 84 and transfection 3.2.3.1.3 Colony PCR 84 3.2.3.1.4 Plasmid DNA isolation 85 3.2.3.1.5 Clone confirmation by double digestion 85 3.2.3.2 Vector construction for M. anisopliae 86 3.2.3.2.1 Cloning Mcl1 gene downstream of β-BUTX 86 Lqq1a gene 3.2.3.2.2 pCAMBIA cloning 87 3.2.3.2.3 E. coli chemical competent cell preparation 87 and transfection 3.2.3.2.4 Colony PCR 87 3.2.3.2.5 Plasmid isolation 88 3.2.3.2.6 Double digestion 88 3.2.4 Protoplast based transformation of B.