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Rubina Mushtaq (M.Phil)

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Rubina Mushtaq (M.Phil) Production of Bacillus thuringiensis Recombinant Cry Proteins and Analysis of Mode of Action of their Toxicity Thesis Submitted to University of the Punjab, Lahore for the Award of Degree of Doctor of Philosophy in Biological Sciences By Rubina Mushtaq (M.Phil) Research Supervisor Prof. Dr. A.R. Shakoori Aizaz-i-Kamal, Tamgha-i-Imtiaz, ECO Laureate Distinguished National Professor, Professor Emeritus School of Biological Sciences, University of the Punjab School of Biological Sciences, University of the Punjab, Lahore, Pakistan 2018 i DEDICATION I would like to dedicate this work to my parents, my husband and to my loving children who helped me unconditionally ii SUMMARY The Cry insecticidal proteins of Bacillus thuringiensis (Bt) are produced by transgenic crops for effective and environmentally-safe insect pest control. These transgenic Bt crops are considered to be the most successful agricultural biotechnology for insect control, yet their sustainability is threatened by the evolution of resistance in targeted pests. The evidence suggests that this resistance is probably due to the alterations in recognition of Cry toxin receptors in the insect midgut membrane. Consequently, information is needed on determinants of receptor recognition for designing improved toxins and to adopt effective insect resistance management practices. In this study four Cry proteins, Cry1Ac, Cry2Ac7, Cry1Fa and Cry1Ie2 were expressed either in E. coli expression system or in native Bt strain. Insoluble Cry1Ac and Cry2Ac7 proteins were refolded and purified prior to use. Bioassays of Cry1Ac (C-terminally truncated version) and Cry2Ac7 protoxins were performed with an armyworm, Spodoptera litura Fabricius (Lepidoptera: Noctuidae), a polyphagous cosmopolitan insect which is a serious crop pest of many Asian countries including Pakistan. Cry2Ac7 was found to be toxic to this pest whereas tnCry1ac (truncated Cry1Ac) was nontoxic. To activate the protoxins, the proteins were trypsin digested and further purified by anion-exchange chromatography. Trypsin activated proteins (toxins) were assayed against velvetbean caterpillar, Anticarsia gemmatalis and soybean looper, Chrysodeixes includens (Pseudoplusia includens) which are lepidopteran pests of crops of great economic importance especially soybean. All these proteins were found to be toxic to both of these pests. Amongst all these toxins Cry1Ac was the most highly toxic protein. In general, C. includens larvae were always less susceptible compared to larvae of A. gemmatalis to these toxins which is in agreement with the previous findings of the relative susceptibilities of these soybean pests for Bt pesticides. The Bt insecticidal proteins Cry1Ac and Cry2Ac7 belong to the three domain family of Bt toxins. Commercial transgenic soybean hybrids produce Cry1Ac to control larvae of the soybean looper and the velvetbean caterpillar. Specificity of Cry proteins is known to be majorly determined by domain II and domain III of the toxin. In this study, we constructed a hybrid toxin (H1.2Ac) containing domains I and II of Cry1Ac and domain III of Cry2Ac7, in an attempt to obtain a protein with enhanced toxicity compared to parent toxins. H1.2Ac protein was expressed in E. coli expression system and was refolded and purified using His- tagged chromatography. Recombinant H1.2Ac protein was also trypsin activated prior to the bioassays. Bioassays with H1.2Ac revealed toxicity to larvae of A. gemmatalis but not to C. iii includens. Saturation binding assays with radiolabeled toxins and midgut brush border membrane vesicles demonstrated no specific H1.2Ac binding to C. includens, while binding to A. gemmatalis was specific and saturable. The competition binding assays showed that Cry1Ac specificity against A. gemmatalis was mainly dictated by domain II. The binding assay in the presence of N-acetylgalactosamine (GalNAc) further clarifies the significance of domain III of Cry1Ac in binding to the receptors of C. includens. Taken together, these distinct interactions with binding sites explain the differential susceptibility of C. includens and A. gemmatalis to Cry1Ac and may provide guidelines for designing the improved toxins against soybean pests. The significance of this work is in identifying Cry2Ac7 as toxic to S. litura and Cry1Ac, Cry2Ac7, Cry1Fa and Cry1Ie2 as toxic to C. includens and A. gemmatalis. Cry1Ie2 can be pyramided with any or all of Cry1Ac, Cry2Ac7 and Cry1Fa proteins in transgenic plants to enhance their efficiency to combat insect pests. While the present study presents evidence for the importance of Cry1Ac domain III for toxicity against C. includens, further research would be needed to identify lethal Cry1Ac receptors and determine their interactions with domains II and/or III of Cry1Ac. This information contributes to the design of more active insecticidal proteins against this pest and our general understanding of the Cry mode of action in Lepidoptera. iv ACKNOWLEDGEMENTS All praise is for Allah Almighty, the most Merciful and the most Beneficent, for giving me inspiration, opportunity and stoutheartedness along this journey. I will be indebted to my thesis advisor, Dr. A. R. Shakoori, for being a kind mentor. His guidance and expertise were invaluable in all aspects of my research and my grooming as a researcher. I am thankful to him for all his precious time, constructive discussions and encouragement. I am highly grateful to Dr. Juan Luis Jurat-Fuentes, University of Tennessee, USA, for providing me the opportunity to work in his laboratory. I acknowledge his patience to understand my questions and obliged for his support and guidance. I am thankful to him for review of my manuscripts and also grateful to his lab members for their help and support. I would like to acknowledge Dr. M. Akhtar, Director General School of Biological Sciences for providing excellent facilities and encouraging atmosphere towards scientific work. I am grateful to Dr. Javed Iqbal for his fatherly kindness for me. I am thankful to Dr. Naeem Rashid who always supported the students and acknowledge his suggestions. I am grateful to all the faculty of SBS for the knowledge they shared with us. My deepest thanks are to all my friends, lab members and SBS fellows. I want to express my gratitude to all those who helped me in any form to complete my work. I do not have words to say thanks to my mother and husband who suffered a lot during my work. Without their support I could not be able to get admission in PhD and complete my research. Special thanks are to my children who overlooked my absence from home and understood my passion for research and granted me permission to live away from them for seven months. Rubina Mushtaq v ABBREVIATIONS NOT DESCRIBED IN THE TEXT Abbreviation Name bp base pair Da Dalton DNA Deoxyribonucleic acid EDTA Ethylene diamine tetra acetic acid FPLC Fast protein liquid chromatography M Molar MgCl2 Magnesium Chloride (NH4)2SO4 Ammonium Sulfate OD Optical density PCR Polymerase chain reaction TEMED N,N,N′,N′-tetramethylethylene-diamine TPCK Tosyl-sulfonyl-phenylalanyl chloromethyl ketone X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside vi CONTENTS Dedication ............................................................................................................. i Summary .............................................................................................................. ii Acknowledgements ............................................................................................ iv Abbreviations not described in the text ............................................................ v List of Figures ..................................................................................................... xi List of tables...................................................................................................... xiv Chapter 1 ............................................................................................................. 1 Introduction and Literature Review .......................................................... 1 Bacillus thuringiensis.................................................................................................. 1 Introduction and discovery ............................................................................... 1 Bt transgenic crops ........................................................................................... 2 Cry protein nomenclature ................................................................................. 4 Crystal protein structure ................................................................................... 5 Domains functions ............................................................................................ 6 Cry-proteins receptors ................................................................................................. 7 Aminopeptidase-N (APN) ................................................................................ 8 Alkaline phosphatase (ALP) ............................................................................ 9 Cadherin-like proteins ...................................................................................... 9 Glycolipids ..................................................................................................... 12 Mode of action of Cry toxins .................................................................................... 12 Pore formation model 1: Formation of pre-pore involves toxin interaction with different receptors in
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