Title: The HAT2 HomeodomainLike Transcription Factor Family: Genes AT5G47370 and AT4G17460
1The HAT2 Homeodomain-Like Transcription Factor
FamilyGenes AT5G47370 and AT4G17460
- Bekah Charney
- HC70AL
- Spring 2006
2What is a HAT2 Homeodomain Transcription Factor?
- Type of transcription factor that is only found
in plants - Has been studied in sunflowers, where it is
expressed primarily in the leaves - When Hahb-4 (sunflower homeobox-leucine zipper
protein) was introduced into Arabidopsis, plants
were more tolerant to water stress conditions - HAT2 gene is induced by auxin, a plant hormone
that regulates growth and development
3Where is AT5G47370 located and what does it code
for?
- AT5G47370 is located on the 5th chromosome
- The gene is 1,598 basepairs in length
- It codes for a HAT2 homeobox leucine zipper
protein - The size of this protein is 284 amino acids
- Within the chromosome, the gene is oriented in
the 3 ? 5 direction
4What are the Anatomical Features of AT5G47370?
_____
_____
______
EXON 1
5URT3
EXON 2
EXON 3
EXON 4
5UTR3
263-413
549-859
943-1022
1119-1598
5Where is AT4G17460 and what does it code for?
- AT4G17460 is on the 4th chromosome
- The gene is 1,467 basepairs in length
- Within the chromosome, the gene is oriented in
the 5 ? 3 direction - It codes for a HAT1 homeobox protein mRNA
- The size of this protein is 283 amino acids
6What are the Anatomical Features of AT4G17460?
_____
_____
______
EXON 1
5URT3
EXON 2
EXON 3
EXON 4
5UTR3
418-746
832-911
1013-1304
183-330
7What Genotypes Did My Plants Reveal?
SALK_055288 knockout for gene AT5G47370
15 Plants were genotyped -8 Homozygous WT -7
Homozygous M -0 Heterozygotes
Wild Type
Homozygous Mutant
Expected WT Size 731 bps Observed WT Size 700
bps Expected Mutant Size 354 bps Observed
Mutant Size 737 and 354 bps
Homozygous mutants were found, showing that a
knockout of this gene does not cause seed
lethality
8Where is the T-DNA Insert in AT5G47370?
LBb1
LBb1
T-DNA
RV
FW
_____
______
EXON 1
5URT
EXON 2
EXON 3
EXON 4
3UTR
- Sequencing results showed that there were two
T-DNA inserts concatomer at nucleotide 1,361,
but oriented in opposite directions - The location of the T-DNA inserts matches SALKs
predictions
9What were the genotypes for SALK_069162 knockout
for gene AT4G17460?
Expected WT Size 1,037 bps Observed WT Size
1,000 bps Expected Mutant Size 669 bps Observed
Mutant Size 784 bps
Since homozygous mutant plants were found, it can
be concluded that a knockout of AT4G17460 does
not cause seed lethality
Wild Type
Homozygous Mutant
10Where is the T-DNA insert in gene AT4G17460?
LBb1
T-DNA
RV
FW
_____
_____
______
EXON 1
5URT
EXON 2
EXON 3
EXON 4
3UTR
- Sequencing results showed that there is a single
T-DNA insert at nucleotide 336 in the first
Intron - This is a 115 basepair difference than what SALK
predicted, which was at nucleotide 451 in the
second Exon
11Where is the gene AT5G47370 active?
My RT-PCR data supports the gene chip data that
this gene is active in both the stem and the
silique, although the levels of activity cannot
be determined
12Where is the gene AT4G17460 active?
My RT-PCR data differs from the gene chip data in
that I found the gene AT4G17460 to be active in
both the inflorescence and the silique
13What work was done with the upstream region of
the AT5G47370 gene and the AT4G17460 gene?
- Amplification of upstream region using iProof
High Fidelity DNA Polymerase - Upstream region ligated with TOPO-vector so lacZ
gene is knocked out - E. Coli cells are transformed with recombinant
plasmids - Restriction Digest with EcoRI
- Plasmids isolated and sequenced with Sp6 and T7
AT5G47370 only
Restriction digest of TOPO-vector with EcoRI
shows a 3.5 kB band and an approximately 2.9 kB
band
Amplified upstream region is 3 kB for gene
AT5G47370
AT4G17460
What further experiments can be done with this
information?
14Did the knockout Arabidopsis plants show any
phenotypic differences than the WT plants?
Knockout for gene AT5G47370
Wild Type
Early Torpedo Stage
NO!
Mature Stage
NO!
15Sterile plants were found in knockout line
SALK_069162
Genotypes of Sterile Plants
Siliques do not fully develop in the sterile
plants
16Wild Type
Knockout for gene AT4G17460
- Some siliques did not develop seeds
- For those that did, seeds did not contain an
embryo
17Conclusion
- A knockout of gene AT4G17460 did not result in
seed lethality, but all known mutants and two
heterozygotes showed sterility - Sterility is simply due to an environmental
factor? - Knockout of gene is causing sterility, either
alone or with other factors?
- A knockout of gene AT5G47370 did not result in
seed lethality or show any phenotypic differences - Gene not important in seed development?
- Another gene is fulfilling its function?
18Whats the next step?
- AT5G47370
- Double knockout
- Use GFPs and cloned upstream region to help
determine where and when gene is being expressed
- AT4G17460
- Check another knockout line (SAIL) to see if
sterile plants result and genotype any sterile
plants - Breed known heterozygotes and check to see if
sterile plants result - Double knockout
- GFPs
19Acknowledgements
I would like to thank Anhthu, Mike, Ria,
Jonathan, Tomo, Brandon, XingJun, Jessica, the
entire HC70AL class and Dr. Goldberg for all
their help and guidance throughout this quarter
I would also specifically like to thank Jennifer,
Ria, Jonathan and XingJun for allowing me to use
their Nomarski photographs in this presentation,
as well as Brandon for the gene chip data used in
this presentation