High throughput genome wide expression analysis of a non-model organism [Elektronische Ressource] : the chickpea root and nodule transcriptome under salt and drought stress / von Carlos Mauricio Molina Medina

Carlos Mauricio Molina Medin

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 33
• Poids de l'ouvrage: 7 Mo

Extrait

High-throughput genome-wide expression analysis of a non-model organism:
The chickpea root and nodule transcriptome under salt and drought stress






Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften




vorgelegt beim Fachbereich Biowissenschaften
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main




von
Carlos Mauricio Molina Medina
aus Kolumbien






Frankfurt am Main
August 2009
(D30)













Vom Fachbereich Biowissenschaften der J.W. Goethe-Universität als Dissertation
angenommen.




















Dekan: Prof. Dr. V. Müller

Gutachter: Professor Dr. Günter Kahl

Pr. Dr. Kurt Weising

Datum der Disputation: 23.01.2009













Este trabajo está especialmente dedicado a mis padres, Carlos y Clara,
y a mi esposa Stefanie por el enorme apoyo que me han brindado en
estos agnos. Gracias por muchas ensenazas de vida, gracias por su
incondicional ayuda







Contents
1 Introduction ........................................................................................... 1
1.1 Chickpea: a world-wide important non-model crop ..................................... 1
1.2 Abiotic stress in plants .................. 4
1.2.1 Drought and salt stress ................................................................................................................. 4
1.2.2 Reactive oxygen species and oxidative stress in plants ................................................................ 6
1.3 Legumes and symbiotic nitrogen fixation ..................... 8
1.3.1 Legume nodules as the nitrogen-fixing organs in roots 8
1.3.2 Legume nodules and abiotic stresses ........................................................................................... 9
1.4 Expression profiling as an important tool in molecular biology ................. 10
1.4.1 Hybridization-based “closed architecture” gene expression profiling ....................................... 11
1.4.2 Sequence-based “open architecture” gene expression profiling ............... 11
1.4.3 SuperSAGE and its application in a non-model organism ........................................................... 12
1.5 Large-scale transcriptome profiling studies of drought- and salt-stressed plants ..................... 14
1.6 Contribution of genome-wide expression profiling and transgenic approaches to the
understanding of water and salt stress in plants ........................................................................ 17
1.6.1 ABA: the most important drought and salt stress signaling hormone in plants ......................... 17
1.6.2 Stress sensing and signal transduction ....................................................................................... 20
1.6.3 The salt overly sensitive pathway for ionic stress in plants ........................ 23
1.7 Aims of the present work ............................................................................................................ 24
1.8 Structure of the present thesis ... 25

2 Materials and methods .......................................................................... 26
2.1 Plant material .............................................................. 26
2.1.1 ICC588: A drought-tolerant chickpea variety .............................................. 26
2.1.2 INRAT-93: A salt-tolerant chickpea variety ................................................. 26
2.1.3 ILC8262: A cold-tolerant chickpea variety .................. 27
2.2 Plant stress treatments ............................................................................... 27
2.2.1 Salt stress treatment ................................................... 28
2.2.2 Drought-stress treatment ........... 29
2.2.3 Cold stress treatment .................................................................................. 30
2.3 Construction of SuperSAGE libraries ........................................................... 31
2.3.1 Total RNA isolation and cDNA synthesis ..................... 31
2.3.2 NlaIII digestion ............................................................................................ 32
2.3.3 cDNA capture with paramagnetic beads and linker ligation ...................................................... 33

2.3.4 Release of linker-tag fragments .................................................................................................. 34
2.3.5 Purification of linker-tag fragments ............................ 35
2.3.6 Filling-in and ditag ligation .......................................................................................................... 35
2.3.7 PCR amplification of ditags ......... 35
2.3.8 Massive ditag amplification and purification for direct sequencing via 454-technology ........... 36
2.4 Data analysis ............................................................................................................................... 36
2.4.1 SuperSAGE tags counting and libraries normalization ................................ 36
2.4.2 Sequence homology alignment of 26-bp SuperTags .. 37
2.4.3 Cluster analysis and functional category distribution analysis of SuperSAGE tags .................... 38
2.5 Confirmatory experiments .......................................................................................................... 38
2.5.1 Rapid amplification of cDNA ends (3’- and 5’-RACE) using UniTags as PCR primers .................. 38
2.5.2 Confirmation of SuperSAGE expression profiles via qRT-PCR ..................................................... 39
2.5.3 Confirmation of expression profiles via UniTags microarray ...................... 40

3 A snapshot of the chickpea transcriptome using SuperSAGE .................. 42
3.1 General aspects of the chickpea SuperSAGE-based transcriptome: Transcripts in very high copy
numbers are not frequent........................................................................................................... 42
3.1.1 Frequencies of tag copy numbers in INRAT-93 SuperSAGE libraries (roots) ............................. 42
3.1.2 Frequencies of tag copy numbers in INRAT-93 SuperSAGE libraries (nodules) ......................... 42
3.1.3 Frequencies of tag copy numbers in ICC588 SuperSAGE libraries (roots) ................................. 42
3.1.4 Frequencies of tag copy numbers in ILC8262 SuperSAGE libraries (leaves) .............................. 43
3.2 UniTag annotation to ESTs deposited in public databases ......................................................... 44
3.3 The resolution of the SuperSAGE technology: Unique transcripts vs transcript-isoforms ......... 45
3.3.1 SuperSAGE and other tagging techniques .................................................................................. 45
3.3.2 SNP-associated alternative tags in chickpea datasets ................................ 47
3.4 Gene-expression changes upon abiotic stresses in chickpea: A large portion of the
transcriptome is stress-responsive ............................................................................................. 47
3.4.1 Salt stress-induced “transcriptome remodelling” in chickpea roots and nodules ..................... 48
3.4.2 Drought stress-induced “transcriptome remodelling” in chickpea roots ................................... 49
3.4.3 Cold stress-induced “transcriptome remodelling” in chickpea leaves ....... 50
3.5 Confirmation of SuperSAGE expression profiles ......................................... 51
3.5.1 Microarray hybridization of spotted SuperSAGE-derived oligos ................................................ 51
3.5.2 Confirmation of SuperSAGE profiles via qRT-PCR ....... 55
3.5.3 Confirmation of UniTags annotation by sequencing of 5’- and 3’-RACE products ..................... 57
3.6 Additional experiments: UniTags conservation between two non-related legumes: chickpea
and lentil ..................................................................................................................................... 59

3.6.1 Description of libraries ................................................................................................................ 59
3.6.2 Common UniTags-proportion related to copy numbers in chickpea and lentil leaves .............. 59

4 The salt stress-responsive transcriptome of chickpea roots ................... 61
4.1 Confirmatory physiological measurements in INRAT-93 ............................................................ 61
4.2 Salt stress-induced differential gene expression in chickpea roots ............................................ 63
4.2.1 Top salt stress-up-regulated UniTags in INRAT-93 roots ............................ 63
4.3 Correlation of SuperSAGE profiles with GO categories in salt-stressed INRAT-93 roots ............ 72
4.3.1 Most over-represented GO biological processes in INRAT-93 salt-stressed roots ..................... 72