Presentation on theme: "The Trihelix Transcription Factor Family Heather Hernandez."— Presentation transcript:
The Trihelix Transcription Factor Family Heather Hernandez
Also called GT factors. DNA binding proteins. Bind to GT elements in promoters. Bind to promoter elements required for light responsiveness. Regulate plant-specific processes. Specific regulatory functions largely unknown. What is a Trihelix Transcription Factor? “Conserved Domain” found in all trihelix proteins.
Trihelix Transcription Factor Genes Gene name: At3g14180 Location: Chromosome 3 Gene size: 1736 Base Pairs Protein size: 444 amino acids Protein encoded: expressed protein similar to 6b-interacting protein 1 (found in Tobacco) UTR REVERSE ORIENTATION 5’3’ EXON
Where in Arabidopsis thaliana is At3g14180 active? RT-PCR 05/23/06 1.5% agarose gel 1.5 hours @ 90v 10 uL EtBr LadderINF RT +INF RT-SIL RT +SIL RT - + Control - Control UCLA Genechip Data + Control - Control
What genotypes are present in my plants? LadderPlant 1Plant 2Plant 3Plant 4Plant 5Wildtype+ Control- Control At3g14180 Genotyping 1% Agarose gel 1.5 hrs @ 105 v 10 uL EtBr T-DNA “bottom” T-DNA “top” Wildtype band LadderPlant 6Plant 7Plant 8Plant 9Plant 10Plant 11Plant 12Plant 13Plant 14Plant 15Plant 16Wildtype+ Control- Control 1% Agarose gel 1.5 hrs @ 105 v 10 uL EtBr At3g14180 Genotyping Wildtype: 1 Heterozygous mutant: 4 Homozygous knockout: 11
Where did the two T-DNA bands insert? Ladder LBb1 LBb1 B T 5’3’ BP 1 BP 1736 830846 Location from SALK matches my sequencing results. (SALK predicted 844 BP into the gene.) My sequencing results:
What is the impact of the T-DNA inserts? No visible phenotypical differences seen in homozygous knockout plants vs. wildtype plants.
What is the impact of the T-DNA inserts on seed development? Wildtype heart stage Mutant heart stage Wildtype late torpedo Mutant torpedo Mutant fully developed Wildtype fully developed suspensor
Trihelix Transcription Factor Gene 2 Gene name: At1g54060 Location: Chromosome 1 Gene size: 1646 Base Pairs Protein size: 384 amino acids Protein encoded: expressed protein similar to 6b-interacting protein 1 (found in Tobacco) UTR 293 BP UTR 200 BP 5’3’ Exon (BP 1-1646) Forward Orientation
Where in Arabidopsis thaliana is At1g54060 active? Ladder INF +RTINF -RTSIL RT +SIL RT -+ Control- Control 1.5 % Agarose gel 1.5 hrs @ 105 v 10 uL EtBr RT-PCR At1g54060Genechip Data
What genotypes are present in my plants? LadderPlant 1Plant 2Plant 3Plant 4Plant 5Plant 6Plant 7Plant 8Plant 9Plant 10Plant 12Plant 13Plant 14Plant 15Wildtype+ Control-Control Genotyping At1g54060 1% Agarose gel 1.5 hrs @ 105 v 10 uL EtBr Wildtype: 6 Heterozygous T-DNA: 3 Homozygous knockout: 5
Where did the T-DNA insert? LBb1 BP 635 5’3’ BP 1BP 1646 My sequencing results: My results matched the SALK institute expected insert site of 640 BP into the gene.
What is the impact of the T-DNA inserts on phenotype? Plant 6 Silique 1 Plant 6, Silique 1, Top Plant 6, Silique 1, Bottom Plant 6, Silique 1, Dissected
How does upstream regulatory activity show where the protein is translated? iProof PCR At3g14180 Ladder“Promoter Region”+ Control- Control 1% Agarose gel 1.5 hrs @ 105 v 10 uL EtBr EcoRI Digest At1g54060 Ladder Sample 1Sample 2Sample 3Sample 4
Acknowledgements Dr. Robert Goldberg Dr. Anthu Bui Mike Gavino Jonathan Russell Garima Yagnik Tomo Kawashima Jessica Luke Dr. Xingjun Wang Lab members!