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.