“ AT5G67300” -AtMYB44, AtMYBr1
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Transcript of “ AT5G67300” -AtMYB44, AtMYBr1
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