Transcript The HAT2 Homeodomain-Like Transcription Factor Family

The HAT2 Homeodomain-Like
Transcription Factor Family:
Genes AT5G47370 and
AT4G17460
Bekah Charney
HC70AL
Spring 2006
What 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
Where 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
What are the Anatomical Features of
AT5G47370?
5’URT3’
EXON 1
263-413
_____
EXON 2
549-859
_____
EXON 3
943-1022
______
EXON 4
1119-1598
5’UTR3’
Where 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
What are the Anatomical Features
of AT4G17460?
5’URT3’
EXON 1
183-330
_____
EXON 2
418-746
_____
EXON 3
832-911
______
EXON 4
1013-1304
5’UTR3’
What 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
Where is the T-DNA Insert in AT5G47370?
LBb1
LBb1
T-DNA
RV
FW
5’URT
EXON 1
EXON 2
_____
EXON 3
______
EXON 4
•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 SALK’s
predictions
3’UTR
What 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
Wild Type
Homozygous
Mutant
Since homozygous mutant plants
were found, it can be concluded
that a knockout of AT4G17460
does not cause seed lethality
Where is the T-DNA insert in gene AT4G17460?
LBb1
T-DNA
RV
FW
5’URT
EXON 1
_____
EXON 2
_____
EXON 3
______
EXON 4
•Sequencing results showed that there is a single TDNA 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
3’UTR
Where 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
Where 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
What 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]
Amplified
upstream
region is 3
kB for gene
AT5G47370
Restriction digest of
TOPO-vector with
EcoRI shows a 3.5 kB
band and an
approximately 2.9 kB
band
AT4G17460
What further experiments can be done with this information?
Did the knockout Arabidopsis plants show any
phenotypic differences than the WT plants?
Wild Type
Knockout for gene AT5G47370
Early Torpedo Stage
NO!
Mature Stage
NO!
Sterile plants were found in knockout line
SALK_069162
Genotypes of Sterile Plants
Number of
Sterile Plants
Siliques do not fully develop in the sterile
plants
Homozygous
WT
0
Heterozygous
2
Homozygous
Mutant
6
Knockout for gene AT4G17460
Wild Type
•Some siliques did not
develop seeds
•For those that did, seeds
did not contain an embryo
Conclusion
• 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?
• 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?
What’s 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
Acknowledgements
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