Arabidopsis thealianaAT3G56230AT4G18650
What are the functions of them?
Are they important for seed development?
Spring 2004 Mariko Onozawa
What is AT3G56230?
・ About 1200bps.
・ 5’UTR.
・ Three SALK lines.
・ This protein containing BTB/POZ domain.
What is AT4G18650?
・ About 1400bps.
・ 5’ and 3’UTR.
・ No SALK line.
・ This protein is related to transcription
factor and has weak similarity to
TGAGG-sequence specific DNA-
binding protein(TGA-2.1).
FV RVFV RV
mRNA
*Find the pattern of mRNA concentration
RT-PCR (SRB)
Genechip database
Gene
* Find KOs of my gene
Madison lines
SALK lines
Phenotype
*Study a phenotype of plants which containing the KO
Check genotypes of the plants (SALK)
mRNA Gene Phenotype
Reverse Genetics
RT-PCR result using SRB (AT3G56230)
Genechip data*RT-PCR result says that the gene which is
homologous toAT3G56230 is expressed at all
stages and organs of SRB.
*Genechip data says that AT3G56230
is expressed only at Mature green seed,
Seedling (3DAI), Roots and stem.
0
500
1000
1500
2000
2500
3000
Ovu
les
24-Hr S
eeds
Mid-
Matu
ration
Matu
re G
S
Post-
Matu
ration G
S
seedlin
g (3DAI)
Leave
s
Roots
Ste
m
AT4G18650AT3G56230
A border between presence and absence
Leaves
+R
T
-RT -RT -RT -RT -RT-RT -RT -RTStem
+
RT
Flow
er +
RT
Ovules
+R
T
Em
bryo +
RT
Axes
+R
T
Leaves
+R
T
Stem
+
RT
control
gene specific
Is there any KO of my gene in Madison lines?*AT3G56230
I couldn’t find any KO of AT3G56230.The insertion site is upstream of the gene. (-594bp)
*AT4G18650
I couldn’t find any KO of AT4G18650.The insertion site is downstream of the gene.
FV RV
R19
FV RV
R15
Checking phenotypes of the plants
T-DNA,homo 1,4WT, homo 2,3,5,6
①WT ①T-DNA ②WT, T-DNA
Only tubilan’s bands
nothing
Some samples don’t have any band.
①WT ①T-DNA②WT, T-DNA
Only tubulin’s bandsNothing
Some seedlings don’t have any band.
③WT, T-DNA
④WT, T-DNAsmears
1 2 3 4 5 6WC 1 2 3 4 5 6WC
③WT, T-DNA
smears
④WT T-DNA
1 2 3 4 5 6 WC 1 2 3 4 5 6 WC
I couldn’t find any significant difference between WT and mutant plants.
T-DNA Wild Type
What should be done next to study the functions?
*Get more plants which have a insertion in the genes.*Observe and study the knockout plants.*Study with other knockout lines.
The final goal is to answer these questions.
*What is the function of the protein?*What is the structure of the protein?*How it is regulated?
What we did in this class is only the beginning of the research.