CtcgaacttgtttttggttcatctctcaaaaccaaaatcactaaagaggagaagattgctaaagtttgataaaacattccaaaatcaATGGCTGATAGGATCAAAGGTCCATGGAGTCCTGAAGAAGACGAGCAGCTTCGTAGGCTTGTTGTTAAATACGGTCCAAGAAACTGGACAGTGATTAGCAAATCTATTCCCGGTAGATCGGGGAAATCGTGTCGTTTACGGTGGTGCAACCAGCTTTCGCCGCAAGTTGAGCATCGGCCGTTTTCGGCTGAGGAAGACGAGACGATCGCACGTGCTCACGCTCAGTTCGGTAATAAATGGGCGACGATTGCTCGTCTTCTCAACGGTCGTACGGACAACGCCGTGAAGAATCACTGGAACTCGACGCTCAAGAGGAAATGCGGCGGTTACGACCATCGGGGTTACGATGGTTCGGAGGATATCGGCCGGTTAAGAGATCGGTGAGTGCGGGATCTCCACCTGTTGTTACTGGGCTTTACATGAGCCCAGGAAGCCCAACTGGATCTGATGTCAGTGATTCAAGTACTATCCCGATATTACCTTCCGTTGAGCTTTTCAAGCCTGTGCCTAGACCTGGTGCTGTTGTGCTACCGCTTCCTATCGAAACGTCGTCTTCTTCCGATGATCCACCGACTTCGTTAAGCTTGTCACTTCCTGGTGCCGACGTAAGCGAGGAGTCAAACCGTAGCCACGAGTCAACGAATATCAACAACACCACTTCGAGCCGCCACAACCACAACAATACGGTGTCGTTTATGCCGTTTAGTGGTGGGTTTAGAGGTGCGATTGAGGAAATGGGGAAGTCTTTTCCCGGTAACGGAGGCGAGTTTATGGCGGTGGTGCAAGAGATGATTAAGGCGGAAGTGAGGAGTTACATGACGGAGATGCAACGGAACAATGGTGGCGGATTCGTCGGAGGATTCATTGATAATGGCATGATTCCGATGAGTCAAATTGGAGTTGGGAGAATCGAGTAGacaaagtgagattattaggaaactgtttaaattggagaagAagaaaaatgctctgtttttttctcctttggattaggcttaagaattttgggttttaaggaaatgtatagaggaaatcgagtgaacaaagctcgagagctggggacgtagtgacgaagacgaagatcaaatttctcttaagctattcaggaaaataaaataaatttttattt

“AT5G67300” -AtMYB44, AtMYBr1

Tomohiro Mochizuki

Junior at Kyoto Univ.

HC70AL S04 Final “Farewell, Thank You, Arigatou, Sayounara” Presentation

AT5G67300 (K8K14.2, ATMYB44, AtMYBr1)Putative myb-related protein

cDNA 1217bp

UTR

UTR

-86 +1 +88 +1005 +1217 +1439

WHAT is AT5G67300?

HOW important is it in embryo development?

HOW is it expressed in Arabidopsis Thaliana?

WHAT happens if you knockout AT5G67300?

WHERE can you find knockout AT5G67300?

HOW can you find the knockout line?

Madison insertion search Round1within 30Superpools

Control

1K

b

1K

b 1 5 10 15 20 25 30

PCR <Fw/LB primers>

PCR <Rv/LB primers>

Madison insertion search Round2within 9 DNA Pools

5 1K

b

98761K

b

4321

Madison insertion;

SUPERPOOL#4

DNAPOOL#34 (7th of SP4)

Between +582 and +583

(right in the middle of the Exon!)

<Fw/LB primers>

4FW583

20FW8114

1FW3114

28FW3014

7RV-1223

7RV-4700 19RV

-1523

17&24FW2914

AT5G67300

19FW6914

13FW7514

AT5G67290 AT5G67320

10RV-4223

AT5G67310

FW RV

018771980

008606-1

0621811247

0837281348

039074270

19RV-423

5’

3’

-5874 -5234 -4268 +1 +89 1026 +1217 +3133 +7090

-5603 -5116 -4175 +1898 +2904 +3247

+2 +1292 +1986 +2920

AT5G67300 situationAT5G67290

FAD dependent oxidoreductase family protein, contains Pfam FAD dependent oxidoreductse.

AT5G67310

Cytochrome P450 family protein.

At5G67320

WD-40 repeat family protein.

5’

3’

?

SALK Genotyping

Sample #1-9 Sample #10-19 Sample #20-29 Sample #30-36

G.S. FW/RV primers

G.S. FW/LB (LBb1) primers

018771

Tubulin control

SALK result

1 17 18

2 3 19 36

4 5 35 34

6 7 33 32

8 31 30

9 10 29 28

11 27 26

12 25 24

13 23 22

21 20

14 15

17

W

WMM

WW W

W WW W

WW WW

WW

W WW

W W

W

WWWWWWWW

WWWW

WWWWW

Mutation homozygous

Wild/Mutation heterozygous

Wildtype homozygous

NO tDNA insertion within AT5G67300 in line#018771

Brandon, call SALK!!!

Checked 36samples of SALK #018771 but……

How is AT5G67300 expressed in SRB?

Lea

f

Ste

m

Flo

wer

Unf

.-ov

ule

14-D

AP

-em

bryo

30-D

AP

ax

isL

eaf

Flo

wer

AT5G67300

Control

<Gene Chip data>

Expressed highly in leaves.

Higher in mature seeds.

<RT-PCR>

mRNA exists in all stages

Arabidopsis Thaliana vs. others

+1 +88 +1005 +1217

173-249 87% 312-408 81%dbEST_ID 16531943

Embryo region: Suspensor

Functional category : 04.1.09 MYB

Protein identification:

(AF122051) tuber-specific and sucrose-responsive element binding factor [Solanum tuberosum]; myb DNA-binding protein (AtMYB73); protein id: At4g37260

UTR

UTR

Solanum tuberosum (Potato) 364a.a.

47% Identity, 56% Positive similarity

tuber-specific and sucrose-responsive element binding factor

Nicotiana Tabacum (tobacco)

329a.a.

62% Identity, 73%Positive similarity

Myb-like protein

HOW can you find the knockout line?

WHERE can you find knockout AT5G67300?

WHAT happens if you knockout AT5G67300?

HOW is it expressed in Arabidopsis Thaliana?

HOW important is it in embryo development?

WHAT is AT5G67300?

By PCR and Southern blotting Pools.

At Madison Facility. Superpool#4, DNAPool#34

Not quite sure.

Mainly in leaves. Post maturation in seeds.

Not very crucial.

SALK Facility. Not reliable. “SALK sometimes gives weird seeds.” Erin Marsh et.al 2004