ERF/AP2 Subfamily A3 and ERF/AP2 Subfamily A6 Genes: AT1G64380 and AT4G39780
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Transcript of ERF/AP2 Subfamily A3 and ERF/AP2 Subfamily A6 Genes: AT1G64380 and AT4G39780
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ERF/AP2 Subfamily A3 and ERF/AP2 Subfamily A6 Genes:
AT1G64380 and AT4G39780
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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
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Where is gene AT1G64380?
AT1G64370
976 bp
AT1G643807,861 bp
1,528 bp742 bp
AT1G64385
2,247 bp
5’ 3’
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Observation of mutant siliques
*Location of T-DNA insert!
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What is gene AT4G39780’s structure?
5’ 3’
Coding Region (819 bp) UTR
256 bp 146 bp
FW
RV
UTR
3’ 5’ orientation
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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
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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
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What are the plant genotypes?
All plants are wild type
Expected WT size: 741 bp
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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
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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
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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.