Tagging Low Temperature Responsive Promoters in Banana Using the Luciferase Reporter Gene

Transcription

1 Laboratory of Tropical Crop Improvement Tagging Low Temperature Responsive Promoters in Banana Using the Luciferase Reporter Gene E. Santos, S. Remy, E. Thiry, S. Windelinckx, R. Swennen and L. Sági KATHOLIEKE UNIVERSITEIT LEUVEN 27 th International Horticultural Congress Aug 13-19, 2006 Seoul, Korea

2 Outline I. Introduction II. III. IV. High-throughput screening for low temperature responsive promoters Isolation and analysis of T-DNA flanking sequences Identification of activated insertion(s) V. Conclusions and perspectives

3 I. Introduction Banana Economy: The 4th most important food crop Diet: Major staple food in tropical countries Characteristics: (Sub)tropical, vegetative propagation, parthenocarpy Genome: Mbp; 11 chromosomes; ploidy: 2x, 3x, 4x; genomic groups (AAA, AAB, ABB ) Molecular tools: Genetic transformation systems, limited sequence data (EST, BAC, SAGE libraries), molecular markers, INIBAP ( Global Musa Genomics Consortium ( Source: INIBAP

4 I. Introduction Promoter usage in transgenic bananas Heterologous Virus Plant Recombinant CaMV35S (derivates), ScBV, TaBV, BSV, BBTV Rice: actin Maize: ubiquitin Arabidopsis: ubiquitin,tubulin Emu, (ocs) 3 mas Homologous Banana: actin, EFE, ACO1, ACC Functionality/stability, less biosafety concerns (cis-genic bananas) Constitutive, tissue specificity, inducible (biotic/abiotic stress)

5 I. Introduction Promoter tagging via Agrobacterium transformation petkul2 T-DNA genomic DNA RB LB luc + -Tnos e35s-neo-t35s P gene Agrobacterium transformation RB LB luc + -Tnos e35s-neo-t35s Screening for different parameters

6 I. Introduction Luciferase reporter gene system luc LUC luciferin + ATP + O 2 oxyluciferin + AMP + PPi + CO 2 + light (562 nm) ultrasensitive digital CCD camera system

7 II. High-throughput screening for low temperature responsive promoters Months after Agrobacterium infection Banana promoter tagging Phase Cell colonies Cell cultures Regenerating cultures Plantlets Culture Screening /Petri Dish 5,600-8,400/image

8 II. High-throughput screening for low temperature responsive promoters Banana - low temperature Low temperature stress chilling / freezing Real-time luciferase screening Cell colonies Plantlets Live Source: 8 C LUC

9 II. High-throughput screening for low temperature responsive promoters Luciferase activation in transgenic cell colonies (~16,000) Screening Treatment Luciferase activity at 26 C No. Frequency (%) Response at 8 C Induced % (No.) Repressed % (No.) 1 RT C 8 C (10) 0.57 (84) LUC Live 26 C 8 C

10 II. High-throughput screening for low temperature responsive promoters Screening during in vitro regeneration Cell colony Live 26 C 8 C Regenerating culture Live 26 C 8 C Plantlet Live 26 C 8 C ET2-17 e35s-luc + (+) (-) Bar = 1cm Low LUC activity High LUC activity

11 III. Isolation and analysis of T-DNA flanking sequences Southern analysis kb MM ET2-lines petkul2 Contr C 5C luc + probe 2.0 Min. No. T-DNA copies: Average: 3.5

12 III. Isolation and analysis of T-DNA flanking sequences TAIL-PCR ET2-17 ET2-34 AD2-1 AD2 AD2-1 AD M bp M bp

13 III. Isolation and analysis of T-DNA flanking sequences Amplification of RB flanking sequences No. of specific PCR amplicons ET2- Line No. T-DNA TAIL-PCR I-PCR copies (Southern) AD2 AD2-1 AD2-5 BsrGI BclI Different sequences NT NT Average NT = Not Tested

14 III. Isolation and analysis of T-DNA flanking sequences Analysis of T-DNA flanking sequences Bioinformatic tools: PlantCARE, PLACE = TATA box = DRE-like = ABRE-like = Transcrition start site (TSSP) 5 -tagged sequence T-DNA 3 -tagged sequence kb kb kb Banana EST 1.17 kb kb 0.86 kb kb kb Direct repeat (306 bp) Direct repeat (681 bp) kb kb

15 IV. Identification of activated insertion(s) RT-PCR RB luc + -Tnos e35s-neo-t35s LB C C Live ET2-17 LUC RB flanking sequence 17-1 M gdna Leaf Leaf Leaf cdna Pseu Corm Root Leaf Pseudostem 17-2 Corm Live ET2-34 LUC C 34 gdna cdna gdna cdna M Leaf Leaf Leaf Leaf Root 34-1

16 V. Conclusions and perspectives Conclusions Different low temperature responses identified Relation low temperature and in vitro regeneration Tagged lines with one to five T-DNA copies Isolation of T-DNA flanking sequences RT-PCR revealed promoter activity Perspectives Back-transformation of selected putative promoter sequences Confirmation of low temperature and developmental regulation

17 Laboratory of Tropical Crop Improvement Acknowledgement VLIR-IUS program cooperation VLIR-ESPOL, Belgium-Ecuador