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’
3’
742 bp
AT1G64370
1,528 bp
2,247 bp
AT1G64380
AT1G64385
7,861 bp
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?
LBb1
T-DNA insert:
300 bp
RV
185 bp
UTR
5’
Coding Region (1,008 bp)
FW
UTR
335 bp
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
3’
Where is gene AT1G64380
expressed?
Gene Chip Data
Expression Levels
Chalazal heart stage
Chalazal seed coat/
linear cotyledon stage
Chalazal seed coat/
Mature green stage
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
What are the possible T-DNA
configurations?
LBb1
T-DNA insert
LBb1
T-DNA insert:
300 bp
RV
185 bp
UTR
Coding Region (1,008 bp)
5’
FW
A concatamer!
UTR
335 bp
Not part of gene
359 bp
3’
What are the possible T-DNA
configurations?
LBb1
T-DNA insert:
300 bp
RV
185 bp
UTR
5’
Coding Region (1,008 bp)
FW
Two inserts located
very close to each
other; sequencing
inconclusive
UTR
335 bp
Not part of gene
359 bp
3’
T-DNA insert
LBb1
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?
Use RT-PCR to detect
AT1G64380 mRNA levels
04/28/09 //120 V
// 1 hour // 1%
agarose
Tubulin control
Genomic DNA
water
g DNA
Silique -RT
Siliqe +RT
Leaf –RT
Leaf +RT
Ladder
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
Negative control
Positive control
Pentr only
PCR to amplify
promoter region
Pentr + gene
Isolate plasmids from
transformed E-coli
No promoter
region detected
Observation of mutant siliques
*Location of T-DNA insert!
What is gene AT4G39780’s structure?
3’ 5’ orientation
RV
256 bp
UTR
Coding Region (819 bp)
5’
146 bp
UTR
3’
FW
Where is the T-DNA insert located?
LBb1
T-DNA insert:
169 bp
RV
256 bp
UTR
5’
Coding Region (819 bp)
FW
Predicted T-DNA insertion site: 695th bp of the gene
WT expected length: 741 bp
T-DNA expected length: 569 bp
146 bp
UTR
3’
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
AT4G39780
*No phenotypic difference
observed in mutant siliques
•No T-DNA inserts found
from screening 24 plants
Look at Arabidopsis
mutant leaves
Check protein production
levels
Obtain different SALK lines
for this gene
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.