Transcript How do we find a knockout for AT4G37790 and what is this

How do we find a
knockout for
AT4G37790 and
what is this gene’s
function in seed
development?
A Research Talk
by Ita Nagy
HC70AL
Spring 2004
What do we know about AT4G37790?
Common Name: HAT-22
Homeobox leucine-zipper
protein
Codes for nuclear protein sized
at 278 AA
Regulatory
DNA binding
Important for transcription factor
function
Contains 1 homeobox domain
Located on chromosome 4 of
Arabidopsis
Forward strand
•THE LEUCINE ZIPPER. Dimers result from
leucine residues at every other turn of the ahelix. When the a-helical regions form a leucine
zipper, the regions beyond the zipper form a Yshaped region that grips the DNA in a scissorslike configuration.
Evolutionary Relationships of HAT
Protein Sequences
-Alignment of HAT9 and
HAT22 reveals 90%
identity in the
homeodomain-leucine
zipper region and 71%
identity overall.
-Little homology is seen
outside of the oustide
the homeodomainleucine zipper region
for other HAT proteins.
Where is HAT-22 active in Arabidopsis
Thaliana and in SRB?
• In SRB, the gene was found
to be the most active in
Sample 1 (Genomic Leaf
+RT), Sample 3 (Genomic
Stem +RT), and Sample A
(Isolated Leaf +RT).
5000
4000
3000
2000
1000
0
wt
Wild Type
Roots
Wild Type
Seedlings
Wild Type
Mature
lec1
Wild Type
24-Hour
• In Arabidopsis, the gene is
most active in lec1 Stem
(signal detection 4087.3 P),
the WT Stem (3170 P), lec1
Leaves (2678.5 P), and WT
Leaves (2491 P).
What is the
structure of
AT4G37790?
•1471 bp, Forward Orientation, 3 exons and 2 introns
•5’ and 3’ region UTR
•FW Primer located 100 bp upstream / RV Primer 540 bp downstream
•Adjacent Genes: AT4G37780 8.9 kb upstream / AT4G37800 6.4 kb
downstream
•Madison t-DNA insert within gene at 661 bp; SALK t-DNA insert outside
gene at –312 bp, perhaps inside the promoter region
Which superpool from the Madison Facility
contains a t-DNA insert in AT4G37790?
Below the control
~800 bp
Superpool 3 contained the correct hit, as shown in the FW
autoradiogram above. The RV autoradiogram contained no
hits. From the gel, the target segment was sized at ~800
bp.
Which DNA pool contains a tDNA insert in AT4G37790?
800 bp
• Amplify PCR products
from 9 DNA pools
belonging to
Superpool 3.
• Use FW & JL202
primers and a +
control, Superpool 3.
• Can we find a PCR
product with the same
size as the hit
segment from our
superpool screening
(~800 bp)?
• The primers amplify
an ~800 bp segment
in DNA Pool 4.
How do SALK Lines help us see how AT4G37790
functions in seed development?
• SALK Line Used:
085964.56.00
• T-DNA Insertion at -312
bp
• Complementary
Orientation
Salk Professor Joanne
Chory with Salk Plants
•Genotype plants through PCR with FW+RV
primers, then t-DNA primer+RV primer because
orientation is complementary. Plants homozygous
for the mutant allele prove that knocking out
AT4G37790 is not embryo lethal. We can
phenotype these plants to see how they are lacking
in development.
What are the genotypes of the SALK
plants with respect to the t-DNA
insert?
1 2 3 4 5 6 7 8 9 wt
There are no homozygous mutants (band in PCR1 and band in PCR2).
Plants 1 and 5 are heterozygous for the mutant allele. They have a band in PCR1 but
also in PCR2 sized at ~2300 bp which is exactly the segment we want, since there are
~2300 bp between the Salk insert at –312 and the RV primer at 2011.
Where do we go from
here?
• SALK LINES: Collect seeds from
heterozygous mutant plants and attempt to
raise homozygous mutants.
• If homozygous mutants are obtained, knockout
is not embryo lethal. However, we can
PHENOTYPE the plants to see whether
abnormalities give us clues to the importance
of AT4G37790 in seed development.
• Salik examination
• MADISON FACILITY: Continue to search for
the knockout by narrowing DNA Pool 4 down
to a specific seed pool and then down to a
specific t-DNA insert line. When this line is
found, grow plants and continue to investigate
whether knocking out AT4G37790 significantly
affects seed development.
How do we Study Gene
Activity
•
In the
SRB?
1.
Isolate RNA from different vegetative organs of
the SRB.
2.
Remove contamination from RNA (such as
genomic DNA).
3.
Perform RT-PCR to transform mRNA into cDNA,
then amplify cDNA using primers for the genespecific region.
4.
RT-PCR Methodology
5.
Run products on a gel to see which samples
amplified. The organs that provided samples
with the most amplification are the ones where
the gene is most active.