Transcript What are the functions of AT3G56230? AT4G18650?

Arabidopsis thealiana
AT3G56230
AT4G18650
What are the functions of them?
Are they important for seed development?
Spring 2004 Mariko Onozawa
What is AT3G56230?
FV
RV
・About 1200bps.
・5’UTR.
・Three SALK lines.
・This protein containing BTB/POZ
domain.
What is AT4G18650?
FV
RV
・About 1400bps.
・5’ and 3’UTR.
・No SALK line.
・This protein is related to transcription
factor and has weak similarity to
TGAGG-sequence specific DNAbinding protein(TGA-2.1).
Reverse Genetics
mRNA
Gene
Phenotype
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)
RT-PCR result using SRB (AT3G56230)
Genechip data
A border between
presence and absence
3000
*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.
AT4G18650
AT3G56230
2500
2000
1500
1000
control
gene specific
500
St e m
Ro o t s
Le ave s
se e dlin g (3 DAI)
Po st - M at u r at io n GS
M at u r e GS
M id- M at u r at io n
2 4 - Hr Se e ds
Ovu le s
-RT
Stem +RT
-RT
Leaves +RT
-RT
Axes +RT
-RT
Embryo +RT
-RT
Ovules +RT
-RT
Flower +RT
-RT
Stem +RT
-RT
Leaves +RT
0
Is there any KO of my gene in Madison lines?
*AT3G56230
R19
FV
RV
I couldn’t find any KO of AT3G56230.
The insertion site is upstream of the gene. (-594bp)
*AT4G18650
FV
I couldn’t find any KO of AT4G18650.
The insertion site is downstream of the gene.
R15
RV
Checking phenotypes of the plants
①WT
①WT
Only tubulin’s bands
①T-DNA
①T-DNA
nothing
Nothing
②WT, T-DNA
②WT, T-DNA
Some seedlings don’t
Some samples don’t
have any band.
have any band.
Only tubilan’s bands
T-DNA,homo 1,4
WT, homo 2,3,5,6
smears
1 2 3 4 5 6 WC ④WT,
1 2 3 4T-DNA
5 6 WC
smears
1 2 3 4 5 6WC 1 2 3 4 5 6WC
④WT
③WT,
T-DNA
③WT,
T-DNA
T-DNA
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.