Evaluation of Approaches for Enhanced Resistance in Plants Against Phloem Limited Pests Shaista Javaid 2017 Department of Biotechnology Pakistan Institute of Engineering and Applied Sciences Nilore, Islamabad, Pakistan This page intentionally left blank. Thesis Approval Form Student’s Name: Shaista Javaid Department: Biotechnology Registration Number: 10-7-1-044-2009 Date of Registration: 20-03-2009 Thesis Title: RECOMMENDATION (if any) by: General comments (attach additional sheet if required) When the final thesis defense of the student has been concluded and all other requirements have been met, I a. Do Recommend that the candidate be certified to the faculty for the degree of Doctor of Philosophy b. Do Recommend that the candidate be certified to the faculty for the degree of Doctor of Philosophy subject to the minor correction in the thesis. c. Do Recommend that the candidate should reappear in the oral defense d. Do NOT Recommend that the candidate be certified to the faculty for the degree of Doctor of Philosophy Examiners Signatures 1. Internal Examiner (Name and Affiliation): 2. Internal Examiner (Name and Affiliation): 3. Internal Examiner (Name and Affiliation): 4. Supervisor (Name and Affiliation): 5. Head of Department (Name): 6. Dean (Research) : Approved by: Head of the Department (Name) :___________________________ Signatures/Date ______________ Distribution: 1. Original to be placed in Student’s Personal file in the office of Dean (Research) 2. Copy to be included in the thesis prior to final submission. Thesis Submission Approval This is to certify that the work contained in this thesis entitled Evaluation of Approaches for Enhanced Resistance in Plants Against Phloem Limited Pests was carried out by Shaista Javaid and in my opinion, it is fully adequate, in scope and quality for the degree of PhD. Furthermore, it is hereby approved for submission for review and thesis defense. Supervisor: _____________________ Name: Dr. Shahid Mansoor (SI) Date: June 30, 2017 Place: NIBGE, Faisalabad. Head, Department of Biotechnology: _____________________ Name: Dr. Shahid Mansoor (SI) Date: June 30, 2017 Place: NIBGE, Faisalabad. Evaluation of Approaches for Enhanced Resistance in Plants Against Phloem Limited Pests Shaista Javaid Submitted in partial fulfillment of the requirements for the degree of PhD. 2017 Department of Biotechnology Pakistan Institute of Engineering and Applied Sciences Nilore, Islamabad, Pakistan Dedications I dedicate all of my work to my beloved Mother and lovely Father. They gave me the confidence to do this job and their prayers make me able to complete the task. ii Declaration of Originality I hereby declare that the work contained in this thesis and the intellectual content of this thesis are the product of my own work. This thesis has not been previously published in any form nor does it contain any verbatim of the published resources which could be treated as infringement of the international copyright law. I also declare that I do understand the terms ‘copyright’ and ‘plagiarism,’ and that in case of any copyright violation or plagiarism found in this work, I will be held fully responsible of the consequences of any such violation. __________________ (Shaista Javaid) June 30, 2017 NIBGE, Faisalabad. iii Copyrights Statement The entire contents of this thesis entitled Evaluation of Approaches for Enhanced Resistance in Plants Against Phloem Limited Pests by Shaista Javaid are an intellectual property of Pakistan Institute of Engineering and Applied Sciences (PIEAS). No portion of the thesis should be reproduced without obtaining explicit permission from PIEAS. iv Table of Contents Declaration of Originality .............................................................................................. ii Copyrights Statement .................................................................................................... iv Table of Contents ........................................................................................................... v List of Figures ............................................................................................................. viii List of Tables ................................................................................................................. x Acknowledgement ........................................................................................................ xi Abstract ...................................................................................................................... xiii List of Publications and Patents .................................................................................. xiv List of Abbreviations and Symbols.............................................................................. xv 1 General Introduction ................................................................................................... 1 1.1 Insecticidal proteins from Bacillus thuringiensis ................................................ 1 1.1.1 Limitations of Bt proteins ............................................................................ 2 1.2 Other insecticidal proteins and their activity ....................................................... 4 1.2.1 Hvt ............................................................................................................... 4 1.2.2 Lectins ......................................................................................................... 6 1.3 Fusion proteins .................................................................................................. 14 1.4 Tissue specific gene expression ........................................................................ 15 1.5 Expression of insecticidal proteins in plants ..................................................... 17 1.5.1 Transient expression .................................................................................. 17 1.5.2 Stable expression of insecticidal proteins in transgenic plants ............. 19 1.6 Need for the project and objectives ................................................................... 20 2 General Methodology ............................................................................................... 21 2.1 Selection of promoter ........................................................................................ 21 2.2 Isolation of insecticidal genes ........................................................................... 21 2.3 Polymerase chain reaction (PCR) ..................................................................... 21 2.4 Cloning of PCR-amplified product ................................................................... 21 2.5 Ligation reaction ............................................................................................... 22 2.6 Preparation of competent cells .......................................................................... 22 2.6.1 Preparation of heat shock E. coli competent cells ..................................... 22 2.6.2 Preparation of electro-competent Agrobacterium tumefaciens cells ......... 22 2.7 Transformation in bacterium ............................................................................. 23 v 2.7.1 Transformation in E. coli ........................................................................... 23 2.7.2 Transformation in Agrobacterium cells ..................................................... 23 2.8 Plasmid isolation ............................................................................................... 23 2.9 Restriction analysis ........................................................................................... 24 2.10 DNA analysis techniques ................................................................................ 24 2.10.1 Agarose gel electrophoresis ..................................................................... 24 2.10.2 Phenol-chloroform precipitation .............................................................. 25 2.11 Plant transformation ........................................................................................ 25 2.12.1 Shifting in soil ......................................................................................... 28 2.13 Confirmation of transgenic plants ................................................................... 28 2.13.1 DNA extraction........................................................................................ 28 2.13.2 RNA extraction ........................................................................................ 29 2.13.3 cDNA Synthesis ...................................................................................... 29 2.14 Insect rearing ................................................................................................... 30 2.14.1 Insect bioassays ....................................................................................... 30 3 Promoter Characterization ........................................................................................ 31 3.1 Introduction ....................................................................................................... 31 3.2 Methodology ..................................................................................................... 33 3.2.1 Isolation and cloning of promoters ............................................................ 33 3.2.2 Plant infiltration ......................................................................................... 35 3.2.3 Histochemical staining assay ....................................................................