Transcript ERF/AP2 Subfamily A3 and ER/AP2 Subfamily A6 Genes
ERF/AP2 Subfamily A3 and ERF/AP2 Subfamily A6 Genes: AT1G64380 and AT4G39780
ERF/AP2 DREB Subfamily
• Characterized by AP2 domain • AP2 family genes-shown to participate in regulation of embryo development • Encodes putative transcription factors (DNA binding motif) • ERF family: CBF and DREB subfamilies – DREB subfamilies contain AP2 and DREB motifs – DREB transcription factors control expression of drought-inducible genes
Where is gene AT1G64380?
976 bp 5’ 742 bp AT1G64370 1,528 bp AT1G64380 7,861 bp 2,247 bp AT1G64385 3’
What is gene AT1G64380’s structure?
185 bp
UTR
5’
Coding Region (1,008 bp) UTR
335 bp
Not part of gene
359 bp 3’
Where is the T-DNA insert located?
185 bp
UTR
5’
Coding Region (1,008 bp) FW LBb1 T-DNA insert: 300 bp UTR
335 bp
RV Not part of gene
359 bp 3’
Predicted T-DNA insertion site: 1,206th bp of the gene WT expected length: 1,195 bp T-DNA expected length: 830 bp
Where is gene AT1G64380 expressed?
Gene Chip Data Chalazal heart stage Chalazal seed coat/ linear cotyledon stage Chalazal seed coat/ Mature green stage Expression Levels
471.6
937.1
406.05
Floral bud/ reproductive Leaf/Vegetative
105.45
What are the plant genotypes?
Homozygous T-DNA (4, 6, 9)
2 T-DNA bands: Top band matches expected amplified T-DNA size (830bp) Lower band approx. 750 bp
Wild type
Expected WT size: 1,195 bp
185 bp
UTR
5’ What are the possible T-DNA configurations?
LBb1 T-DNA insert LBb1 T-DNA insert: 300 bp Coding Region (1,008 bp) FW UTR
335 bp
RV Not part of gene
359 bp 3’
A concatamer!
185 bp
UTR
5’ What are the possible T-DNA configurations?
LBb1 T-DNA insert: 300 bp Coding Region (1,008 bp) FW UTR
335 bp
RV Not part of gene
359 bp 3’
Two inserts located very close to each other; sequencing inconclusive T-DNA insert LBb1
What do these results mean?
Wildtype (WT/WT) 7 Hemizygous (WT/T-DNA) 0 Homozygous (T-DNA/T-DNA) 3 Presence of homozygous mutant plants:
Knockout of gene AT1G64380 does NOT lead to seed lethality
Where in
Arabidopsis Thaliana
is gene AT1G64380 transcribed?
Use RT-PCR to detect AT1G64380 mRNA levels
Tubulin control Genomic DNA 04/28/09 //120 V // 1 hour // 1% agarose
Gene AT1G64380 is expressed in both Arabidopsis leaf and silique tissues; supports Gene Chip data
How can we visualize where gene AT1G64380 is transcribed?
Promoter Cloning -no recombinant plasmids found in 42 bacteria colonies screened
ASCI restriction digest and PCR colony screening Expected size of recombinant: 4.3 kb
WHY? Initial ligation was not successful
Isolate plasmids from transformed E-coli
PCR to amplify promoter region
No promoter region detected
Observation of mutant siliques
*Location of T-DNA insert!
What is gene AT4G39780’s structure?
3’ 5’ orientation 5’ 256 bp
UTR RV Coding Region (819 bp)
146 bp
UTR
3’
FW
Where is the T-DNA insert located?
5’
LBb1 T-DNA insert: 169 bp
256 bp
UTR Coding Region (819 bp) RV
146 bp
UTR FW
3’
Predicted T-DNA insertion site: 695th bp of the gene WT expected length: 741 bp T-DNA expected length: 569 bp
Where is gene AT4G39780 expressed?
Gene Chip Data Chalazal seed coat/ globular stage Floral Bud/ reproductive General Seed Coat/ Globular Stage General Seed coat/ Heart stage General seedcoat/ pre-globular stage Leaf/Vegetative
What are the plant genotypes?
All plants are wild type Expected WT size: 741 bp
What do these results mean?
Enough plants were screened
T-DNA was not inserted into the gene
Fatality of knockout to seed development is inconclusive No further analysis can be done on these plants
Future Research
AT1G64380
*No phenotypic difference observed in mutant siliques
Look at
Arabidopsis
mutant leaves Check protein production levels Obtain different SALK lines for this gene AT4G39780 •
No T-DNA inserts found from screening 24 plants
Obtain a different SALK line for this gene --Repeat previous methods
Acknowledgements
Thank you to Anhthu, Kristen, Daisy, Brandon, Min, Tomo, Kelli, Ingrid and Dr. Goldberg for making this lab experience happen!! This has been an incredible quarter.