ERF/AP2 Subfamily A3 and ERF/AP2 Subfamily A6 Genes:

AT1G64380 and AT4G39780

ERF/AP2 DREB Subfamily

• Characterized by AP2 domain• AP2 family genes-shown to participate in

regulation of embryo development• Encodes putative transcription factors (DNA

binding motif)• ERF family: CBF and DREB subfamilies

– DREB subfamilies contain AP2 and DREB motifs– DREB transcription factors control expression of

drought-inducible genes

Where is gene AT1G64380?

AT1G64370

976 bp

AT1G643807,861 bp

1,528 bp742 bp

AT1G64385

2,247 bp

5’ 3’

What is gene AT1G64380’s structure?

5’ 3’

UTR Coding Region (1,008 bp) UTR

185 bp

335 bp

Not part of gene

359 bp

Where is the T-DNA insert located?

T-DNA insert:

300 bp

5’ 3’

UTR Coding Region (1,008 bp) UTR

185 bp

335 bpFW

RV

LBb1

Predicted T-DNA insertion site: 1,206th bp of the gene

WT expected length: 1,195 bp

T-DNA expected length: 830 bp

Not part of gene

359 bp

Where is gene AT1G64380 expressed?

Chalazal heart stage

Chalazal seed coat/ linear cotyledon stage

Chalazal seed coat/ Mature green stage

Expression Levels

471.6

937.1

406.05

105.45Leaf/Vegetative

Floral bud/ reproductive

Gene Chip Data

What are the plant genotypes?

Homozygous T-DNA (4, 6, 9)

2 T-DNA bands:

Top band matches expected amplified T-DNA size (830bp)

Lower band approx. 750 bp

Wild type

Expected WT size: 1,195 bp

What are the possible T-DNA configurations?

300 bp

5’ 3’

UTR Coding Region (1,008 bp) UTR

185 bp

335 bpFW

RV

LBb1

Not part of gene

LBb1

A concatamer!

T-DNA insert:T-DNA insert

359 bp

What are the possible T-DNA configurations?

300 bp

5’ 3’

UTR Coding Region (1,008 bp) UTR

185 bp

335 bpFW

RV

LBb1

Not part of gene

LBb1

T-DNA insert:

T-DNA insert

359 bp

Two inserts located very close to each other; sequencing inconclusive

What do these results mean?

Wildtype

(WT/WT)

Hemizygous

(WT/T-DNA)

Homozygous

(T-DNA/T-DNA)

7 0 3

Presence of homozygous mutant plants:

Knockout of gene AT1G64380 does NOT lead to seed lethality

Where in Arabidopsis Thaliana is gene AT1G64380 transcribed?

water

g D

NA

Siliq

ue -R

TS

iliqe +

RT

Leaf –R

TL

eaf +R

TL

add

er

04/28/09 //120 V // 1 hour // 1% agarose

Use RT-PCR to detect AT1G64380 mRNA levels

Tubulin control

Genomic DNA

Gene AT1G64380 is expressed in both Arabidopsis leaf and silique tissues; supports Gene Chip data

How can we visualize where gene AT1G64380 is transcribed?

Promoter Cloning -no recombinant plasmidsfound in 42 bacteria

coloniesscreened

ASCI restriction digest and PCR colony screening

WHY?

Initial ligation was not successful

Isolate plasmids from transformed E-coli

PCR to amplify promoter region

Expected size of recombinant: 4.3 kb

No promoter region detected

Neg

ative con

trol

Po

sitive con

trol

Pen

tr on

ly

Pen

tr + g

ene

Observation of mutant siliques

*Location of T-DNA insert!

What is gene AT4G39780’s structure?

5’ 3’

Coding Region (819 bp) UTR

256 bp 146 bp

FW

RV

UTR

3’ 5’ orientation

Where is the T-DNA insert located?

T-DNA insert:

LBb1

Predicted T-DNA insertion site: 695th bp of the gene

WT expected length: 741 bp

T-DNA expected length: 569 bp

5’ 3’

Coding Region (819 bp) UTR

256 bp 146 bp

FW

RV

UTR

169 bp

Where is gene AT4G39780 expressed?

Chalazal seed coat/ globular stage

Floral Bud/ reproductive

General Seed Coat/ Globular Stage

Leaf/Vegetative

General Seed coat/ Heart stage

Gene Chip Data

General seedcoat/ pre-globular stage

What are the plant genotypes?

All plants are wild type

Expected WT size: 741 bp

What do these results mean?

Enough plants were screened

T-DNA was not inserted into the gene

Fatality of knockout to seed development is inconclusive

No further analysis can be done on these plants

Future Research

AT1G64380*No phenotypic difference

observed in mutant siliques

Look at Arabidopsis

mutant leaves

Check protein production levels

Obtain different SALK lines

for this gene

AT4G39780•No T-DNA inserts found from screening 24 plants

Obtain a different SALK line for this gene

--Repeat previous methods

Acknowledgements

Thank you to Anhthu, Kristen, Daisy, Brandon, Min, Tomo, Kelli, Ingrid and Dr. Goldberg for making this lab experience happen!! This has been an incredible quarter.