High Throughput Search of Drought Tolerant Genes in Agave sisalana L. SANIA RIAZ CENTRE OF EXCELLENCE IN MOLECULAR BIOLOGY UNIVERSITY OF THE PUNJAB LAHORE PAKISTAN (2015) High Throughput Search of Drought Tolerant Genes in Agave sisalana L. A THESIS SUBMITTED TO UNIVERSITY OF THE PUNJAB IN FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MOLECULAR BIOLOGY By SANIA RIAZ Supervisor: Dr. Tayyab Husnain (Prof & Acting Director) Centre of Excellence in Molecular Biology. University of the Punjab, Lahore CERTIFICATE It is certified that the research work described in this thesis is the original work of the author Ms. Sania Riaz and has been carried out under my direct supervision. I have personally gone through all the data reported in the manuscript and certify their correctness and authenticity. It is further certified that the material included in this thesis have not been used in part or full manuscript already submitted or in the process of submission in partial/complete fulfillment of the award of any other degree from any other institution. It is also certified that the thesis has been prepared under my supervision according to the prescribed format and we endorse its evaluation for the award of Ph.D degree through the official procedures of the university. In accordance with the rules of the centre, data book #852 is declared as unexpendable document that will be kept in the registry of the Centre for a minimum of three years from the date of the Thesis defense examination. Signature of the supervisor________________________________ Name: Dr. Tayyab Husnain Designation: Prof & Acting Director (Allah) Most Gracious! It is He Who has taught the Qur'an. He has created man: He has taught him speech (and Intelligence). The sun and the moon follow courses (exactly) computed; and the herbs and the trees- both (alike) bow in adoration. And the Firmament has He raised high, and He has set up the balance (of Justice), in order that you may not transgress (due) balance. It is He Who has spread out the earth for (His) creatures: There in is fruit and date-palms, producing spathes (enclosing dates): Also corn with (its) leaves and stalks for fodder and sweet- smelling plants. Then which of the favours of your Lord will you deny? (SURAH Rehman, AYAH 1-13) DEDICATED TO My BELOVED PARENTS ACKNOWLEDGEMENT I am extremely grateful to almighty “ALLAH” whose bountiful blessings enabled me to complete this research project. He bestowed us with his "HOLY QURAN", and prophet “MUHAMMAD” (Peace be upon him), who enlightens the hearts of us in our lives. I wish to acknowledge several key figures that contributed much to my research endeavor. A journey is easier when you travel together. Interdependence is certainly more valuable than independence. This thesis is the result of six years of work whereby I have been accompanied and supported by many people. It is a pleasant aspect that I have now the opportunity to express my gratitude for all of them. First and foremost I would like to take this opportunity to place on record my deep sense of gratitude to my supervisor, Dr. Tayyab Husnain (Prof & Acting Director, CEMB). I have been in his project since January 2010 when I started my PhD research work. During this whole period of six years I have known him as a sympathetic and principle-centered person. His overly enthusiasm and integral view on research and his mission for providing „only high quality work and not less‟, has made a deep impression on me. I owe him lots of gratitude for having me shown this way of research. Especially the extensive comments and the many discussions and the interactions with him had a direct impact on the final form and quality of this thesis. He could not even realize how much I have learned from him. Besides of being an excellent supervisor, I am really glad that I have come to get know Dr. Tayyab Husnain as a brilliant teacher and hardworking supervisor. I am greatly indebted to my Lab Incharge Dr. Bushra Rashid (Assistant Prof), for her sympathetic attitude and cordial co-operation throughout the progress of this research. Cordial thanks are due to Dr. Ahmad Ali Shahid (Associate Prof.) & Dr. Abdul Qayyum (Assistant Prof) who made it a convivial place to work. They all kept an eye on the progress of my work and always were available when I needed their advice and valuable suggestions. I am also obliged to pay my sincere thanks to all my lab colleagues especially Beenish Aftab, who helped me in difficult circumstances to accomplish this project. It was a great pleasure to work with all of them. I can‟t express my feelings about the Higher Education Commission (HEC), who provided me such opportunity to make my dreams true. Without HEC 5000 Indigenous Ph.D fellowship scheme this opportunity would mere a dream for me. I am also very much thankful to almighty ALLAH Pak Who granted me the most beautiful and sweetest feelings of life and for which a mother can wish for. I was blessed with a daughter Hareem Ayesha and a son Abdul Hadi during the journey of my doctorate studies. Last but not the least, I wish to pay my sincerest gratitude to my parents Muhammad Riaz and Jahan Ara Riaz, who always remembered me in their prayers and raised me with a love of science and supported me in all pursuits and of course this is the sweetest outcome of their blessings. I am highly indebted and obliged to thank my better half Mr. Rizwan-ur-Rehman, a Ph.D scholar in Tianjin university of science and Technology,China. He has always supported and encouraged me to do my best in all matters of life. I wish to thank my affectionate brothers Irfan Afzal, Sami Riaz, Yasir Riaz, Babar Riaz and Hassan Riaz and my dearest sisters Shazia Irfan and Dr. Sadaf Abdul Rauf. They all have always supported and encouraged me to do my best in all matters of life. I will simply say thank you very much to all of you. Sania Riaz i CONTENTS Table of Contents i List of Figures vi List of Tables ix Abbreviations xi Summary xiv 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 5 2.1 HISTORY AND ORIGIN OF AGAVE 5 2.2 TAXONAMY AND CLASSIFICATION OF AGAVE 5 2.3 CRASSULACEAN ACID METABOLISM 6 2.4 LIFE SPAN OF AGAVE SPECIES 7 2.5 PHARMACOLOGICAL AND ETHNO MEDICINAL PROPERTIES 8 OF AGAVE PLANTS 2.6 EFFECT OF LOW AND HIGH TEMPERATURES ON AGAVE 9 2.7 MOLECULAR STUDIES ON AGAVE PLANTS 10 2.8 HIGH THROUGHPUT SEARCH OF UPREGULATED GENES IN AGAVE SISALANA L. 10 2.9 ROLE OF ABIOTIC STRESSES ON AGRICULTURAL CROPS AND THEIR PRODUCTION 11 2.10 PLANTS DEFENCE AGAINST ABIOTIC STRESSES 13 2.11 COMPLEXITY OF ABIOTIC STRESS SIGNALING 14 2.12 SENSORS INITIATE MULTIPLE SIGNALING PATHWAYS 15 2.13 SIGNAL TRANSDUCTION SIGNALING PATHWAYS 15 2.14 OXIDATIVE STRESS SIGNALING UNDER ABIOTIC STRESSES 16 2.15 ROLE OF LEA TYPE GENES UNDER ABIOTIC STRESSES 17 2.16 ROLE OF SOS PATHWAYS AND ACTIVATION OF 18 HOMEOSTASIS 2.17 POSSIBLE FATE OF SIGNALING PATHWAYS 18 2.18 GENE EXPRESSION REGULATION BY TRANSCRIPTION 19 FACTORS (TFS) 2.19 ZINC FINGER (ZNF) TRANSCRIPTION FACTORS 21 ii 2.20 ROLE OF bZIP TFS IN STRESS TOLERANCE IN PLANTS 22 2.21 ROLE OF WRKY TFs and Cys2/His2 ZINC FINGER PROTEINS 22 UNDER OSMOTIC STRESS 2.22 ROLE OF AP2/ERF, MYB AND bHLH TF‟s IN STRESSED 23 ENVIRONMENT ADAPTATION 2.23 GENE EXPRESSION TRIGGERED BY NAC TRANSCRIPTION 23 FACTOR 2.24 DROUGHT STRESS INFLUENCES ON GAS EXCHANGE 24 PARAMETERS 2.25 EFFECTS OF DROUGHT STRESS ON PLANT WATER RELATIONS 27 2.26 ROLE OF BIOCHEMICAL MARKERS IN RESPONSE TO 27 DROUGHT STRESS 2.27 ROLE OF MOLECULAR BIOLOGY IN PRESENT ERA 31 3 MATERIALS AND METHODS 33 3.1 PLANT MATERIAL AND DROUGHT TREATMENT 33 3.2 DETERMINATION OF FIELD CAPACITY 34 3.3 MICROSCOPIC EXAMINATION OF LEAF EPIDERMAL TISSUE 35 3.4 PLANTS‟ PHYSIOLOGICAL ANALYSIS UNDER DROUGHT STRESS 35 3.5 WATER RELATED ATTRIBUTES AND LEAF SURFACE AREA 37 35 3.5.1 Leaf Relative Water Content (LRWC) 35 3.5.2 Leaf Surface Area 36 3.6 PLANTS‟ BIOCHEMICAL ANALYSIS UNDER DROUGHT STRESS 36 3.6.1 Proline Content 36 3.6.2 Lipid Peroxidation Assay (Malondialdehyde Content) 37 3.6.3 Total Chlorophyll Content 37 3.7 STATISTICAL ANALYSIS 37 3.8 TOTAL RNA ISOLATION 38 3.8.1 Agarose Gel Electrophoresis 38 3.8.2 Quantification of Total RNA 39 3.8.3 DNase Treatment 39 3.9 CONSTRUCTION OF cDNA LIBRARY 39 3.9.1 Isolation of mRNA from Total RNA 39 3.9.2 Precipitation of mRNA 40 iii 3.9.3 Double Strand cDNA Construction 40 3.9.4 Ligating the attB1 Adapter 42 3.9.5 cDNA Size Selection 43 3.9.6 Gel Elution 43 3.9.7 BP Recombination Reaction 43 3.10 SCREENING OF COLONIES ON MEDIA 45 3.10.1 Plasmid Isolation 45 3.10.2 Restriction Analysis of Cloned cDNA 46 3.11 PREPARATION OF cDNA MICROARRAY PLATFORM 46 3.11.1 Clone Pickling and Culturing 46 3.11.2 PCR Amplification of Inserts 46 3.11.3 Purification of PCR products prior to sequencing 47 3.12 CLONE SEQUENCING 48 3.13 REMOVAL OF VECTOR SEQUENCES USING VEC SCREEN 49 3.14 BLAST SEARCH 49 3.15 GENE ONTOLOGY (GO) AND FUNCTIONAL ANNOTATION 49 3.16 PRINTING OF cDNA MICROARRAY 49 3.17 HYBRIDIZATION OF TARGET WITH cDNA MICROARRAY 51 PLATFORM 3.17.1 Target Preperation 51 3.17.1.1 Aminoallyl Labeling 51 3.17.1.2 Removal of Un-Incorporated aa-dUTP and free amines 52 3.17.1.3 Coupling aa-cDNA to Cyanine Dyeester 52 3.17.1.4 Removal of Uncoupled Dye 52 3.17.2 Hybridization 53 3.17.2.1 Pre-Hybridization 53 3.17.2.2 Hybridization 53 3.17.2.3 Slide Washing 53 3.17.3 Slide Scanning 53 3.18 IMAGES PROCESSING AND RAW DATA GENERATION 54 3.19 DATA NORMALIZATION AND ANALYSIS 54 3.19.1 Data Normalization 54 3.19.2 The Midas Project 54 iv 3.19.3 Tm4 Mev Analysis 54 3.20 SEQUENCING OF DIFFERENTIALY EXPRESSED TRANSCRIPTS 55 3.21 VALIDATION STUDIES BY QUANTITATIVE REAL-TIME PCR 55 3.22 BIOINFORMATIC STUDIES 55 3.22.1 Blast Search 55 3.22.2.