STACKING OF UPSTREAM REGULATORY GENES TO CONFER ABIOTIC STRESS TOLERANCE ...

323 Pages·2015·11.42 MB·English

STACKING OF UPSTREAM REGULATORY GENES
TO CONFER ABIOTIC STRESS TOLERANCE IN
RICE (Oryza sativa L.)

PATIL MAHESH
PAL 0039

DEPARTMENT OF CROP PHYSIOLOGY
UNIVERSITY OF AGRICULTURAL SCIENCES
BENGALURU
2015
STACKING OF UPSTREAM REGULATORY GENES
TO CONFER ABIOTIC STRESS TOLERANCE IN
RICE (Oryza sativa L.)

PATIL MAHESH
PAL 0039

Thesis submitted to the
UNIVERSITY OF AGRICULTURAL SCIENCES, BANGALORE
in partial fulfilment of the requirements
for the award of the degree
DOCTOR OF PHILOSOPHY
in
CROP PHYSIOLOGY

BENGALURU DECEMBER, 2015
Affectionately
Dedicated to
My Beloved Parents
Shivagouda and
Shanta

ACKNOWLEDGEMENT
This is certainly a good opportunity for me to thank a number of people who have made
this journey possible. Firstly, Dr. P. Chandrashekara Reddy, Professor, Department of
Crop Physiology, UAS, Bengaluru thanks for your unconditional guidance and advises
throughout this PhD Programme. More importantly, thank you for allowing me to grow
both professionally and personally.
I feel immense pleasure in expressing my profound sense of gratitude and indebtedness
to Dr. M. Udayakumar, Project co-ordinator and Dr. T. G. Prasad, Associate Project
co-ordiantor, Department of Crop Physiology, UAS, GKVK, Bengaluru, members of
advisory committee for their valuable guidance, constant supervision and
encouragement throughout the period of my study and also providing financial assistance
throughout my study.
I also express my sincere gratitude and thanks to the other members of my advisory
committee Dr. N. Nataraja Karaba, Professor, Department of Crop Physiology, UAS,
Bengaluru, Dr. Anitha Peter, Associate Professor, Department of Plant Biotechnology,
UAS, Bengaluru, Dr. M. P. Rajanna, Rice Breeder, ZARA, VC Farm, Mandya, for
their advice and constant encouragement during the course of the study.
I thank all the teaching staff of the Department of Crop Physiology for their best co-
operation and timely help to carry out my research program successfully. I also thank
Dr. Rama for initial guidance and support, Aruna, Preethi Reddy and Shweta for their
care, encouragement and support in conducting experiments.
I would like to thank my parents who are the Almighty’s most treasured gift to me. I feel
scanty of words to magnitude the boundless love and tireless sacrifice and affection
showed on me by my father Shivagouda and my mother Shanta whom I wish to idolize
throughout my life for making me what I am today. My brother Mahantesh and sister-in -
law Deepa for their love and care.
I would like to thank my senior friends Jalendra, Dr. Ramu, Dr. Babitha, Dr. Pruthvi,
Dr. Rajashekara Reddy, Sajeevan, Dr. Nagarjuna, Dr. Vasanta, for their care, constant
encouragement and support during my research program. I also cherish the beautiful
moments spent with all my friends, Parvathi, Pushpa, Dr. Kabeya, Dr. Raju,
Dr. Mahesh Salimath, Madhura, Nisarga, Rashmi, Jyothi, Amar, Ashwini, Pavithra,
Pratiba, Dr. Mati, Sashi, Appanna, Dhanya, Sapna. Kavya, Nitin, Nagangouda,
Ramesh,
I would like to acknowledge the financial support from Department of
Biotechnology, HUB programme and Niche Area of Excellence, ICAR, to support my
Ph.D. programme. Sincere thanks to all my senior and junior friends of Department of
Crop Physiology for making my stay a memorable.

Bengaluru
December, 2015 Patil Mahesh
STACKING OF UPSTREAM REGULATORY GENES TO CONFER
ABIOTIC STRESS TOLERANCE IN RICE (Oryza sativa L.)
PATIL MAHESH
THESIS ABSTRACT
Rice cultivation under aerobic condition has phenomenal relevance in terms of saving
water. But reduced water availability and high VPD affecting crop growth and
productivity. Thus it is important to improve adaptation of rice under aerobic condition
by improving water relations and cellular level tolerance (CLT) mechanisms. Improving
CLT to sustain cell metabolic activities has relevance under stress and among several
CLT mechanisms, protein synthesis and stability are crucial. From this context, an
attempt has been made to validate three regulatory genes i.e. Pg47 (Pennisetum glaucum
47, a RNA helicase), OseIF4E (Oryza sativa translational initiation factor 4E) and
PgHSF4 (Pennisetum glaucum heat shock factor 4) involved in protein synthesis and
protection in a model system tobacco. Transgenics expressing single and multigene
cassette showed higher tolerance to diverse stresses viz. salinity, ER, heavy metal,
drought and osmotic stress, besides showing enhanced expression of few stress
responsive genes compared to wild type under stress. However, lines expressing
multigene cassette showed superior phenotype and tolerance over lines expressing single
genes with significantly higher protein content and lesser malondialdehyde level. This
demonstrated the significance of co-expressing more than one stress specific
genes/TFs over single gene. To improve adaptation of rice under semi-irrigated aerobic
conditions rice transgenics co-expressing multigene cassette were developed in the
background of AC39020 genotype having superior water relations. True transformants
were identified based on glyphosate tolerance. The transgenics showed improved
tolerance to salinity, ER and heavy metal stress apart from drought stress. Under
drought stress, many promising transgenic lines showed reduced spikelet sterility and
higher yield compared to wild type. The study provides proof of concept that
maintaining efficient translation and protein protection improves CLT and transgenic
is a potential option to combine or pyramid the relevant traits to improve field level
tolerance.

December, 2015
Department of Crop Physiology (P. Chandrashekar Reddy)
UAS, GKVK, Bengaluru Chairman
ಭತ್ತದಲ್ಲಿ ಅಜೈವಿಕ ಒತ್ತಡ ಸಹಿಷ್ಣುತೆಯನ್ಣು ಹೆಚ್ಚಿಸಲಣ ನಿರ್ದಿಷ್ಟವಾದ ಅನ್ಣವಂಶಿಕ
ಧಾತ್ಣಗಳ ಸಾಮೂಹಿಕ ಅಭಿವಯಕತನೆ

ಪಾಟೀಲ್ ಮಹೆೀಶ್

ಪ್ರಬಂಧದ ಸಾರಾಂಶ

ಅರೆನಿೀರಾವರಿ ಪ್ದಧತಿಯಣ ಕಡಿಮೆ ನಿೀರಿನ್ಲ್ಲಿ ಭತ್ತವನ್ಣು ಬೆಳೆಯಣವ ಪ್ರಮಣಖ ಪ್ದಧತಿಯಾಗಿದೆ. ಸಾಮಾನ್ಯವಾಗಿ
ಇಲ್ಲಿ ಬೆಳೆದ ಭತ್ತವು ಅನೆೀಕ ಅಜೈವಿಕ ಒತ್ತಡಗಳಿಗೆ ಸಿಲಣಕಿ ಸಂಭಾವಯ ಉತ್ಾಾದಕತೆಗಿಂತ್ ಅತಿ ಕಡಿಮೆ ಇಳಣವರಿ
,
ನಿೀಡಣತ್ತದೆ. ಈ ಅಜೈವಿಕ ಒತ್ತಡಗಳಿಗೆ ಸಹಿಷ್ಣುತೆಯನ್ಣು ವೃರ್ದಧಸಲಣ ಆಣ್ವಿಕ ಸಹಿೀಷ್ಣುತೆಯನ್ಣು
ಹೆಚ್ಚಿಸಣವುದರ ಜೂತೆಗೆ ( ಅಂಗಾಶದಲ್ಲಿನ್ ನಿೀರಿನ್ ಪ್ರಮಾಣವನ್ಣು
cellular level tolerance)
ಕಾಪಾಡಿಕೂಳಳಬೆೀಕಣ. ಆಣ್ವಿಕ ಸಹಿಷ್ಣುತೆಯಲ್ಲಿ, ಪ್ರರಟೀನ್ ಉತ್ಾಾದನೆ ಮತ್ಣತ ಸಿಿರತೆಯಣ ಅತಿ ಮಣಖಯವಾದ
ಪಾತ್ರವನ್ಣು ವಹಿಸಣತ್ತವೆ. ಈ ನಿಟಟನ್ಲ್ಲಿ ಅಜೈವಿಕ ಒತ್ತಡಗಳಲ್ಲಿ ಪ್ರರಟೀನ್ ಉತ್ಾಾದನೆಯನ್ಣು ಹೆಚ್ಚಿಸಲಣ
ಪಿ.ಜಿ-೪೭ ( ) ಮತ್ಣತ ಒ.ಎಸ್.ಇ.ಐ.ಎಫ್-೪ಇ (
Pennisetum glaucum 47, a RNA helicase Oryza sativa
) ಗಳನ್ಣು ಆಯ್ಕೆಮಾಡಲಾಯಿತ್ಣ ಹಾಗೂ ಉತ್ಾಾರ್ದತ್ ಪ್ರರಟೀನಿನ್
translation initiation factor 4E
ಸಿಿರತೆಯನ್ಣು ಕಾಪಾಡಲಣ ಪಿ.ಜಿ.ಎಚ್,ಎಸ್,ಎಫ್-೪’ ನ್ಣು ( )
Pennisetum glaucum heat shock factor 4
ಆಯ್ಕೆಮಾಡಲಾಯಿತ್ಣ. ಈ ಮೂರಣ ಧಾತ್ಣಗಳನ್ಣು ತ್ಂಬಾಕಣ ತ್ಳಿ ಕ-೩೨೬ ಯಲ್ಲಿ ಬಿಡಿಬಿಡಿಯಾಗಿ ಮತ್ಣತ
ಸಾಮೂಹಿಕವಾಗಿ, ಜೈವಿಕ ತ್ಂತ್ರಜ್ಾುನ್ ಬಳಸಿ ಅಭಿವಯಕತಪ್ಡಿಸಲಾಯಿತ್ಣ. ಮಾಪ್ಿಡಿತ್ ಸಾಲಣಗಳಣ ಅಜೈವಿಕ
ಒತ್ತಡಗಳಾದ ಕ್ಷಾರತೆ, ಇ.ಅರ್ ( ), ಭಾರಿಲೂೀಹ ಮತ್ಣತ ಬರ
Endoplasmic Reticulum, ER stress
ಪ್ರಿಸಿಿತಿಯಲ್ಲಿ ಹೆಚ್ಚಿನ್ ಸಹಿಷ್ಣುತೆ ಮತ್ಣತ ಬೆಳವಣ್ವಗೆಯನ್ಣು ತೊೀರಿದವು. ಪ್ರತೆಯೀಕ ಧಾತ್ಣಗಳನ್ಣು ಹೊಂರ್ದದ
ಕಣಲಾಂತ್ರಿಗಳಿಗಿಂತ್, ಮೂರಣ ಧಾತ್ಣಗಳನೊುಳಗೊಂಡ ( ) ಕಣಲಾಂತ್ರಿಗಳಣ ಹೆಚ್ಚಿನ್ ಒತ್ತಡ
multigene
ಸಹಿಷ್ಣುತೆ, ಪ್ರರಟೀನ್ ಉತ್ಾಾದನೆ ಮತ್ಣತ ಕಡಿಮೆ ಪ್ರಮಾಣವನ್ಣು ತೊೀರಿದವು. ಪ್ರಿಣಾಮವಾಗಿ, ಈ
MDA
ಮೂರಣ ಧಾತ್ಣಗಳನೊುಳಗೊಂಡ ಧಾತ್ಣವಾಹಕವನ್ಣು ( ಆಯದ ಭತ್ತದ ತ್ಳಿಯಾದ ಎಸಿ-
multigene construct)
೩೯೦೨೦’ಗೆ ಜೈವಿಕ ತ್ಂತ್ರಜ್ಾುನ್ರ್ದಂದ ಅಭಿವಯಕತಪ್ಡಿಸಲಾಯಿತ್ಣ. ಕಣಲಾಂತ್ರಿಗಳಣ ಕ್ಷಾರತೆ, ಇ.ಅರ್ ( ),
ER stress
ಭಾರಿಲೂೀಹ ಮತ್ಣತ ಬರ ಪ್ರಿಸಿಿತಿಯಲ್ಲಿ ಉತ್ತಮವಾದ ಸಹಿಷ್ಣುತೆ ಮತ್ಣತ ಬೆಳವಣ್ವಗೆಯನ್ಣು ತೊೀರಿದವು.
ಇದರ ಜೂತೆಗೆ ಆಯದ ಕಣಲಾಂತ್ರಿ ಸಾಲಣಗಳಣ ಬರ ಪ್ರಿಸಿಿತಿಯಲ್ಲಿ ಮೂಲತ್ಳಿಗಳಿಗಿಂತ್ ಹೆಚ್ಚಿನ್ ಕಾಳಣ ಕಟ್ಣಟವಿಕ
ಮತ್ಣತ ಇಳಣವರಿಯನ್ಣು ತೊೀರಿದವು. ಒಟ್ಾಟರೆ ಪ್ರತೆೀಕ ಅಭಿವಯಕತನೆಗಿಂತ್ ಸಾಮೂಹಿಕವಾಗಿ ಹೆಚ್ಚಿನ್ ಗಣಣಗಳನ್ಣು

ಅಭಿವಯಕಿತಸಣವದರಿಂದ ಅನೆೀಕ ಅಜೈವಿಕ ಒತ್ತಡಗಳಿಗೆ ಸಹಿೀಷ್ಣುತೆಯನ್ಣು ಒದಗಿಸಬಹಣದೆಂಬ ಪ್ರಿಕಲಾನೆಗೆ ಈ
ಅಧಯಯನ್ವು ಯಶಸಿಿಯಾಗಿ ಪ್ುರಾವೆಯನ್ಣು ನಿೀಡಿದೆ.

ಡಿಸಂಬರ ೨೦೧೫
,
ಬೆಳೆ ಶರಿೀರ ಕಿರಯಾಶಾಸರ ವಿಭಾಗ .
ಪಿ ಚಂದ್ರಶ ೇಕರ್ ರ ಡ್ಡಿ
ಕೃಷಿ ವಿಶಿವಿದ್ಾಯನಿಲಯ ಜಿ.ಕ.ವಿ.ಕ ಬೆಂಗಳೂರಣ- ೫೬೦೦೬೫
, , ಮುಖ್ಯಸಲಹ ಗಾರರ
CONTENTS

CHAPTER TITLE PAGE No.
I INTRODUCTION 1-6
II REVIEW OF LITERATURE 7-42
III MATERIAL AND METHODS 43-66
IV RESULTS 67-113
V DISCUSSION 114-135
VI SUMMARY 136-141
VII REFERENCES 142-182
APPENDICES 183-196
LIST OF TABLES
Table Page
Title
No. No.
1 Helicases up-regulated under various abiotic stresses 28
2 Validated HSF genes associated with stress tolerance 39
Segregation analysis of T tobacco transgenic plants expressing
3 1 75-76
PgHSF4
Segregation analysis of T tobacco transgenic plants expressing
4 1 80-81
OseIF4E
Segregation analysis of T tobacco transgenic plants expressing
5 1 84-85
Pg47
Segregation analysis of T tobacco transgenic plants expressing
6 1 88-89
multigene construct
7 Yield and biomass analysis of pot grown T2 transgenic plants 103
Productivity analysis of T transgenic and wild type plants grown
2
8 in contained field under moisture regime I (MR-I) and moisture 103
regime II (MR-II) conditions
Mean yield and biomass of selected transgenic lines grown in pot
9 and contained field conditions (MR-I & MR-II) and also seedling 106
growth in NaCl and DTT stress experiment
Productivity analysis of transgenic and wild type plants under
10 109
contained field facility in two moisture regimes
List of selected transgenic lines showing yield, biomass and
11 111
spikelet fertility under MR-I and MR-II conditions in T generation
3