Differential Proteomic Analysis for Identifying
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DIFFERENTIAL PROTEOMIC ANALYSIS FOR IDENTIFYING PROTEINS INVOLVED IN THE BIOSYNTHESIS OF PICROSIDE−I AND PICROSIDE−II FROM Picrorhiza kurroa−AN ENDANGERED MEDICINAL HERB Thesis submitted in fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY By AMIT SUD Department of Biotechnology and Bioinformatics JAYPEE UNIVERSITY OF INFORMATION TECHNOLOGY WAKNAGHAT, DISTRICT SOLAN, H.P., INDIA JANUARY 2018 I Copyright @ JAYPEE UNIVERSITY OF INFORMATION TECHNOLOGY WAKNAGHAT JANUARY 2018 ALL RIGHTS RESERVED II TABLE OF CONTENTS Content Page No. INNER FIRST PAGE I DECLARATION VII CERTIFICATE VIII ACKNOWLEDGEMENT X – XI ABSTRACT XII – XIII LIST OF FIGURES XIV – XVI LIST OF TABLES XVII LIST OF ABBREVIATIONS XVIII – XX CHAPTER 1: INTRODUCTION 1 – 11 CHAPTER 2: REVIEW OF LITERATURE 12 – 48 2.1 INTRODUCTION 12 2.2 Picrorhiza Kurroa 18 2.3 BOTANICAL CLASSIFICATION AND MORPHOLOGY OF P. 18 kurroa 2.4 HISTORY OF P. kurroa 19 2.5 GEOGRAPHICAL DISTRIBUTION OF P. kurroa 20 2.6 VERNACULAR NAMES 23 2.7 MEDICINAL PROPERTIES OF P. kurroa 23 2.7.1. HEPATOPROTECTIVE 23 2.7.2. ANTI-TUMOR/ANTI-NEOPLASTIC ACTIVITY 24 2.7.3. ANTIOXIDANT 24 2.7.4. IMMUNOMODULATORY/ ADJUVANT 24 2.7.5. ANTIASTHMATIC 24 2.7.6. VIRAL HEPATITIS 25 2.7.7. ANTI-INFLAMMATORY 25 2.7.8. ANTI-ALLERGIC AND ANTI-PHYLACTIC 25 2.7.9. ANTI-CHOLESTATIC 26 2.7.10. POTENTIAL DRUG AGAINST NAFLD 26 III 2.8 MEDICINAL PROPERTIES OF PURE CHEMICAL 27 CONSTITUENTS OF P. kurroa 2.8.1. PICROSIDE-I 27 2.8.2. PICROSIDE-II 27 2.8.3. APOCYANIN 28 2.8.4. CATALPOL 28 2.9 PHYTOCHEMISTRY OF P. kurroa – AN OCEAN OF 29 PHYTOCHEMICAL CONSTITUENTS 2.9.1. CUCURBITACINS 33 2.9.2. IRIDOIDS 34 2.10 FACTORS AFFECTING THE BIOSYNTHESIS AND 38 ACCUMULATION OF CHEMICAL CONSTITUENTS IN P. kurroa 2.10.1. VARIATIONS IN P-I AND P-II LEVELS IN RELATION 38 TO TISSUE AGE IN P. kurroa 2.10.2. EFFECTS OF DIFFERENT 39 GROWTH/DEVELOPMENTAL STAGES OF P. kurroa 2.10.3. EFFECTS OF ENVIRONMENTAL FACTORS ON P-I 40 AND P-II LEVELS IN P. kurroa 2.10.3.1. EFFECT OF HEIGHT AND TEMPERATURE 40 ON PICROSIDE CONTENT 2.10.3.2. EFFECT OF ALTITUDE ON PICROSIDES 41 CONTENT 2.11 MOLECULAR BASIS OF PICROSIDES BIOSYNTHESIS 42 CHAPTER 3: MATERIALS AND METHODS 49 - 65 3.1 PLANT SAMPLES 49 3.1.1 FOR DIFFERENTIAL PROTEIN EXPRESSION 49 STUDIES IN P. kurroa TO IDENTIFY PROTEINS INVOLVED IN THE BIOSYNTHESIS OF PICROSIDE-I AND INCREASED SHOOT BIOMASS 3.1.2 FOR DIFFERENTIAL PROTEIN EXPRESSION 50 STUDIES IN P. kurroa TO INVESTIGATE THE PRESENCE OF PROTEINS INVOLVED IN THE IV BIOSYNTHESIS OF PICROSIDE-I AND PICROSIDE-II IN STOLONS 3.2 OPTIMIZATION OF PROTEIN EXTRACTION METHOD 50 3.2.1 TRIS-CL EXTRACTION METHOD 50 3.2.2 PHOSPHATE BUFFER EXTRACTION METHOD 50 3.2.3 TCA-ACETONE EXTRACTION METHOD 50 3.2.4 WATER EXTRACTION METHOD 51 3.3 PROTEIN ESTIMATION 51 3.3.1 LOWRY’S PROTEIN ESTIMATION 51 3.3.2 BIO-RAD RC DC PROTEIN ASSAY 52 3.4 PROTEIN EXTRACTION 54 3.4.1 FOR DIFFERENTIAL PROTEIN EXPRESSION 54 STUDIES IN P. kurroa TO IDENTIFY PROTEINS INVOLVED IN THE BIOSYNTHESIS OF PICROSIDE-I AND INCREASED SHOOT BIOMASS 3.4.2 FOR DIFFERENTIAL PROTEIN EXPRESSION 55 STUDIES IN P. kurroa TO INVESTIGATE THE PRESENCE OF PROTEINS INVOLVED IN THE BIOSYNTHESIS OF PICROSIDE-I AND PICROSIDE-II IN STOLONS 3.5 PROTEIN SEPARATION 55 3.5.1 FOR DIFFERENTIAL PROTEIN EXPRESSION 55 STUDIES IN P. kurroa TO IDENTIFY PROTEINS INVOLVED IN THE BIOSYNTHESIS OF PICROSIDE-I AND INCREASED SHOOT BIOMASS 3.5.2 FOR DIFFERENTIAL PROTEIN EXPRESSION 60 STUDIES IN P. kurroa TO INVESTIGATE THE PRESENCE OF PROTEINS INVOLVED IN THE BIOSYNTHESIS OF PICROSIDE-I AND PICROSIDE-II IN STOLONS 3.6 GEL STAINING 60 3.7 IMAGE ACQUISITION AND ANALYSIS 62 3.7.1 FOR SDS-PAGE GELS 62 V 3.7.2 FOR 2-D GEL ELECTROPHORESIS GELS 62 3.8 MALDI-TOF MS ANALYSIS 62 3.9 MASCOT PROTEIN IDENTIFICATION 63 3.10 WESTERN BLOT ANALYSIS 64 3.10.1 SDS-PAGE 64 3.10.2 PROTEIN TRANSFER 64 3.10.3 IMMUNOBLOTTING 64 3.10.4 IMAGE ACQUISITION AND ANALYSIS 65 CHAPTER 4: RESULTS 66 - 119 4.1 OPTIMIZATION OF PROTEIN EXTRACTION METHOD 66 4.2 DIFFERENTIAL PROTEIN EXPRESSION STUDIES IN P. kurroa 68 TO IDENTIFY PROTEINS INVOLVED IN THE BIOSYNTHESIS OF PICROSIDE-I AND INCREASED SHOOT BIOMASS 4.3 WESTERN BLOT ANALYSIS 111 4.4 DIFFERENTIAL PROTEIN EXPRESSION STUDIES IN P. kurroa 111 TO INVESTIGATE THE PRESENCE OF PROTEINS INVOLVED IN THE BIOSYNTHESIS OF PICROSIDE-I AND PICROSIDE-II IN STOLONS CHAPTER 5: DISCUSSION 121 - 142 5.1. OPTIMIZATION OF PROTEIN EXTRACTION METHOD 121 5.2. DIFFERENTIAL PROTEIN EXPRESSION STUDIES IN P. 121 KURROA TO IDENTIFY PROTEINS INVOLVED IN THE BIOSYNTHESIS OF PICROSIDE-I AND INCREASED SHOOT BIOMASS 137 5.3. DIFFERENTIAL PROTEIN EXPRESSION STUDIES IN P. KURROA TO INVESTIGATE THE PRESENCE OF PROTEINS INVOLVED IN THE BIOSYNTHESIS OF PICROSIDE-I AND PICROSIDE-II IN STOLONS SUMMARY 143 – 144 CONCLUSION AND FUTURE PROSPECTS 145 REFERENCES 146 LIST OF PUBLICATIONS 180 VI DECLARATION I hereby declare that the work reported in the Ph.D. thesis entitled “Differential proteomic analysis for identifying proteins involved in the biosynthesis of picroside−I and picroside−II from Picrorhiza kurroa−An endangered medicinal herb” submitted at Jaypee University of Information Technology, Waknaghat, India, is an authentic of my work carried out under the supervision of Dr. Hemant Sood and co-supervision of Dr. Chanderdeep Tandon. I have not submitted this work elsewhere for any other degree or diploma. I am fully responsible for the contents of my Ph.D. Thesis Amit Sud Enrollment No. 106567 Department of Biotechnology and Bioinformatics Jaypee University of Information Technology, Waknaghat, India Date: VII CERTIFICATE This is to certify that the work reported in the Ph.D. thesis entitled “Differential proteomic analysis for identifying proteins involved in the biosynthesis of picroside−I and picroside−II from Picrorhiza kurroa−An endangered medicinal herb” which is being submitted by Amit Sud (Enrollment No. 106567) in fulfillment for the award of degree of Doctor of Philosophy in Biotechnology at Jaypee University of Information Technology, Waknaghat, India is a bonafide record of his original work carried out under my supervision. This work has not been submitted partially or wholly to any other University or Institute for the award of this or any other degree or diploma. Dr. Hemant Sood Dr. Chanderdeep Tandon (Supervisor) (Co-Supervisor) Associate Professor Director Department of Biotechnology & Bioinformatics Amity Institute of Biotechnology Jaypee University of Information Technology Amity University Waknaghat, India Noida, India Date: Date: VIII Dedicated to my loving Parents & Brothers for their endless support IX ACKNOWLEDGEMENT The realization of a research enterprise such as a Doctor of Philosophy is never solely the result of hard work but is aided by many discussions and good teamwork. Support and encouragement come from different sources in various ways. A very tight schedule and tremendous help from a great number of people have helped me to complete this endeavour. In this acknowledgment, I would like to gratefully thank all those persons who have contributed to making this effort a success. First, I am extremely grateful to my research guide, Dr Hemant Sood, Associate Professor, Department of Biotechnology and Bioinformatics for her valuable guidance, scholarly inputs and consistent encouragement I received throughout the research work. Thank you, Madam, for all your help and support. I would also like to thank my co- supervisor Dr Chanderdeep Tandon, Professor, and Director, Amity Institute of biotechnology, Amity University, Noida for his guidance and support throughout my doctorate research and during the completion of this thesis. This feat was possible only because of the unconditional support provided by Sir. A person with an amicable and positive disposition, Sir has always made himself available to clarify my doubts despite his busy schedules and I consider it as a great opportunity to do my doctoral programme under his guidance and to learn from his research expertise. His positive attitude and infinite motivation kept me going. I will never forget his inspirational stories which he used to tell me to stay motivated. In short, he is the coolest Professor to work with. Thank you, sir, for your never-ending support. I would also like to express my heart felt gratitude to Dr. Simran Tandon Madam for her help through my tough times. Without her support, completing my Ph. D would have been a lot more difficult. I thank Dr Rajinder Singh Chauhan, Ex-Dean, & Ex-HOD, and Dr. Sudhir Syal current acting HOD, Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, for their constructive comments, for their important support during this work and finally for setting up good standards of research in the whole department. I would like to thank JUIT administration, Prof. (Dr.) Vinod Kumar, Vice Chancellor, JUIT; Maj Gen (Retd.) Rakesh Bassi, Registrar and Dean of students JUIT; Prof. Samir Dev Gupta, Dean (Academic & Research); Prof. (Dr.) T. S. Lamba, Ex-Dean (Academic & Research) and Dr Yajulu Medury (Ex-COO, Jaypee Education System) for X providing an opportunity to pursue a Doctorate Degree and advanced lab infrastructure to achieve this scientific endeavor of my life. I am thankful to the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi for providing financial support. I am also thankful to Dr Jitendraa Vashistt, Dr. Gunjan Goel and Dr. Sunil Kumar Khah for their help and valuable suggestions throughout my research work. They were always ready to help me whenever I approached them with my queries.