THE FUNCTION OFAT5G04410 (NAC2)

ANDAT3G10500 (NAC2-like)

NAC FamilyYUYA HIRAI

A Cartoon Structure of My First Gene　 (At5g04410)

exon1

5’ 3’3257

5’UTR 3’UTR

intron

•　 6 exons and 5 introns •　 3257bp•　 Forward orientation•　 No apical meristem(NAM) family protein•　 567aa

In What Organs Is the At5g04410 Gene Being Transcribed?

The picture of RT-PCR

This gene is being transcribed in both leaf

and silique.

Confirm the expected size band

WT

Leaf

+R

TW

T le

af +

RT

WT

leaf

-RT

WT

siliq

ue +

RT

WT

siliq

ue -R

T

gDN

A (p

os.)

Wat

er (n

eg.)

Is this gene important for the seed development?

To answer this question,search for plants having

T-DNA inserted to this gene.

Tubulin(440bp)

200bp280bp

The Location Site of T-DNA

exon1

5’ 3’3257

5’UTR

3’UTR

intron

Fw primer-100

Rv primer T-DNA

LBb1

583

SALK_025098

Reverse orientation

Location site 583bp

Wild-Type band - 3.2 kbp

T-DNA band - 0.8 kbp

3100

Which Plant Have T-DNA Inserted In the At4g04410 Gene?

Fig.1 Fig.2

Fig.2Fig.4 Genotyping(Fig.2)

Wild type: 7 plants

Homo: 3 plants

Hetero: 4 plants

Excise the marked band and sequence Expected result!

The size of T-DNA band is about 0.8kbp Expected result!

1 122 3 4 5 6 7 8 9 10 11 13 14 Isolation of gDNA from 14 plants (Fig.1)gDNA

RNA

WT(3.2kbp)

T-DNA(0.8kbp)

Nomarski Images (At5g04410)

Wild type

Homozygous mutant

There is no difference in phenotypes between wild type and mutant!

Walking stick

Torpedo

Walking stick

globular

exon

1 2419

5’UTR

A Cartoon Structure of My Second Gene (At3g10500)

5’’ 3’

•　 6 exons and 5 introns•　 2419bp•　 Forward orientation•　 No apical meristem 　 (NAM) family protein•　 549aa

intron

3’UTR

In What Organs Is the At3g10500 Gene Being Transcribed?

Confirm the expected size band

This gene also is being transcribed in both leaf and

silique!

Is this gene important for the seed development?

To answer this question, Search for plants having T-DNA inserted

to this gene

WT

leaf

+R

T

WT

siliq

ue -R

T

WT

leaf

-RT

WT

siliq

ue +

RT

Leaf

(fro

m T

A)

Seed

(from

TA

)gD

NA

(pos

.)

Wat

er(n

eg.)

WT

leaf

+R

TW

T le

af -R

TW

T si

lique

+R

TW

T si

lique

-RT

gDN

A(p

os.)

Wat

er(n

eg.)

0.4kbpExpectedsize+Tubulin -Tubulin

exon

1 2419

38

2407

Fw primer

Rv primer

5’UTR

The Location Site of T-DNA5’’ 3’

intron

3’UTR

T-DNA

2189

LBb1SALK_018311

Reverse orientation

Location site 2189bp

Wild-Type band - 2.4kbp

T-DNA band - 2.2kbp

Which Plants Have T-DNA Inserted In the At3g10500 Gene?

Genotyping (Fig.2)

Wild type: 1 plant

Homo: 7 plants

Hetero: 6 plants

The size of T-DNA band is about 0.3kbp Not expected result!

Excise the marked band and sequence

Very interesting result!

1 2 3 4 5 6 7 8 9 10 11 12 13 14Fig.1

Fig.21 2 3 4 5 6 7 88 9 10 11 12 13 14 Po

s.N

eg.

Isolation of gDNA from 14 plants (Fig.1)

gDNA

RNA

WT(2.3kbp)

T-DNA(0.3kbp)

exon

1 2419

38

2407

Fw

Rv

5’UTR

The Location Site of T-DNA From My Experiment

5’’ 3’

intron

3’UTR

T-DNA

2189

LBb1

SALK_018311

Reverse orientation

Location site 2189bp

LBb1

Forward orientation

There was no differences in phenotypes between WT and mutant plants!

Nomarski Images (At3g10500)

Wild type

Homozygous mutant

globular

globular

torpedo

torpedo

The Suggestion of My ExperimentThere was no differences in phenotype between wild type and mutant plants both At5g04410 and At3g10500.

The possibility that one gene compensates the function of another gene!

More experiments need to be done…

Cross At5g04410 mutant with At3g10500 mutant and check the phenotype of the double knockout mutant!

Thanks everyone especially…Dr Goldberg

and Kunio!!! (of course Tomo!!)

yu_yan_yu_yan_yu_yan@yahoo.co.jp