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?
AT1G64370
976 bp
AT1G643807,861 bp
1,528 bp742 bp
AT1G64385
2,247 bp
5’ 3’
What is gene AT1G64380’s structure?
5’ 3’
UTR Coding Region (1,008 bp) UTR
185 bp
335 bp
Not part of gene
359 bp
Where is the T-DNA insert located?
T-DNA insert:
300 bp
5’ 3’
UTR Coding Region (1,008 bp) UTR
185 bp
335 bpFW
RV
LBb1
Predicted T-DNA insertion site: 1,206th bp of the gene
WT expected length: 1,195 bp
T-DNA expected length: 830 bp
Not part of gene
359 bp
Where is gene AT1G64380 expressed?
Chalazal heart stage
Chalazal seed coat/ linear cotyledon stage
Chalazal seed coat/ Mature green stage
Expression Levels
471.6
937.1
406.05
105.45Leaf/Vegetative
Floral bud/ reproductive
Gene Chip Data
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
What are the possible T-DNA configurations?
300 bp
5’ 3’
UTR Coding Region (1,008 bp) UTR
185 bp
335 bpFW
RV
LBb1
Not part of gene
LBb1
A concatamer!
T-DNA insert:T-DNA insert
359 bp
What are the possible T-DNA configurations?
300 bp
5’ 3’
UTR Coding Region (1,008 bp) UTR
185 bp
335 bpFW
RV
LBb1
Not part of gene
LBb1
T-DNA insert:
T-DNA insert
359 bp
Two inserts located very close to each other; sequencing inconclusive
What do these results mean?
Wildtype
(WT/WT)
Hemizygous
(WT/T-DNA)
Homozygous
(T-DNA/T-DNA)
7 0 3
Presence of homozygous mutant plants:
Knockout of gene AT1G64380 does NOT lead to seed lethality
Where in Arabidopsis Thaliana is gene AT1G64380 transcribed?
water
g D
NA
Siliq
ue -R
TS
iliqe +
RT
Leaf –R
TL
eaf +R
TL
add
er
04/28/09 //120 V // 1 hour // 1% agarose
Use RT-PCR to detect AT1G64380 mRNA levels
Tubulin control
Genomic DNA
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 plasmidsfound in 42 bacteria
coloniesscreened
ASCI restriction digest and PCR colony screening
WHY?
Initial ligation was not successful
Isolate plasmids from transformed E-coli
PCR to amplify promoter region
Expected size of recombinant: 4.3 kb
No promoter region detected
Neg
ative con
trol
Po
sitive con
trol
Pen
tr on
ly
Pen
tr + g
ene
Observation of mutant siliques
*Location of T-DNA insert!
What is gene AT4G39780’s structure?
5’ 3’
Coding Region (819 bp) UTR
256 bp 146 bp
FW
RV
UTR
3’ 5’ orientation
Where is the T-DNA insert located?
T-DNA insert:
LBb1
Predicted T-DNA insertion site: 695th bp of the gene
WT expected length: 741 bp
T-DNA expected length: 569 bp
5’ 3’
Coding Region (819 bp) UTR
256 bp 146 bp
FW
RV
UTR
169 bp
Where is gene AT4G39780 expressed?
Chalazal seed coat/ globular stage
Floral Bud/ reproductive
General Seed Coat/ Globular Stage
Leaf/Vegetative
General Seed coat/ Heart stage
Gene Chip Data
General seedcoat/ pre-globular stage
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.