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“ AT5G67300” -AtMYB44, AtMYBr1

“ AT5G67300” -AtMYB44, AtMYBr1. HC70AL S04 Final “Farewell, Thank You, Arigatou, Sayounara” Presentation. Tomohiro Mochizuki Junior at Kyoto Univ. Ctcgaacttgtttttggttcatctctcaaaaccaaaatcactaaagaggagaagattgctaaagtttgataaaacattccaaaatca

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“ AT5G67300” -AtMYB44, AtMYBr1

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  1. “AT5G67300” -AtMYB44, AtMYBr1 HC70AL S04 Final “Farewell, Thank You, Arigatou, Sayounara” Presentation TomohiroMochizuki Junior at Kyoto Univ. Ctcgaacttgtttttggttcatctctcaaaaccaaaatcactaaagaggagaagattgctaaagtttgataaaacattccaaaatca ATGGCTGATAGGATCAAAGGTCCATGGAGTCCTGAAGAAGACGAGCAGCTTCGTAGGCTTGTTGTTAAATACGGTCCAAGAAACTGG ACAGTGATTAGCAAATCTATTCCCGGTAGATCGGGGAAATCGTGTCGTTTACGGTGGTGCAACCAGCTTTCGCCGCAAGTTGAGCAT CGGCCGTTTTCGGCTGAGGAAGACGAGACGATCGCACGTGCTCACGCTCAGTTCGGTAATAAATGGGCGACGATTGCTCGTCTTCTC AACGGTCGTACGGACAACGCCGTGAAGAATCACTGGAACTCGACGCTCAAGAGGAAATGCGGCGGTTACGACCATCGGGGTTACGAT GGTTCGGAGGATATCGGCCGGTTAAGAGATCGGTGAGTGCGGGATCTCCACCTGTTGTTACTGGGCTTTACATGAGCCCAGGAAGCC CAACTGGATCTGATGTCAGTGATTCAAGTACTATCCCGATATTACCTTCCGTTGAGCTTTTCAAGCCTGTGCCTAGACCTGGTGCTG TTGTGCTACCGCTTCCTATCGAAACGTCGTCTTCTTCCGATGATCCACCGACTTCGTTAAGCTTGTCACTTCCTGGTGCCGACGTAA GCGAGGAGTCAAACCGTAGCCACGAGTCAACGAATATCAACAACACCACTTCGAGCCGCCACAACCACAACAATACGGTGTCGTTTA TGCCGTTTAGTGGTGGGTTTAGAGGTGCGATTGAGGAAATGGGGAAGTCTTTTCCCGGTAACGGAGGCGAGTTTATGGCGGTGGTGC AAGAGATGATTAAGGCGGAAGTGAGGAGTTACATGACGGAGATGCAACGGAACAATGGTGGCGGATTCGTCGGAGGATTCATTGATA ATGGCATGATTCCGATGAGTCAAATTGGAGTTGGGAGAATCGAGTAGacaaagtgagattattaggaaactgtttaaattggagaag Aagaaaaatgctctgtttttttctcctttggattaggcttaagaattttgggttttaaggaaatgtatagaggaaatcgagtgaaca aagctcgagagctggggacgtagtgacgaagacgaagatcaaatttctcttaagctattcaggaaaataaaataaatttttattt

  2. AT5G67300 (K8K14.2, ATMYB44, AtMYBr1)Putative myb-related proteincDNA 1217bp U T R U T R -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?

  3. Madison insertion search Round1within 30Superpools PCR <Fw/LB primers> Control 1Kb 1Kb 1 5 10 15 20 25 30 PCR <Rv/LB primers>

  4. Madison insertion search Round2within 9 DNA Pools <Fw/LB primers> Madison insertion; SUPERPOOL#4 DNAPOOL#34 (7th of SP4) Between +582 and +583 (right in the middle of the Exon!) 5 6 1Kb 1 2 3 4 7 8 9 1Kb

  5. AT5G67300 situation AT5G67290 FAD dependent oxidoreductase family protein, contains Pfam FAD dependent oxidoreductse. ? 008606 -1 At5G67320 WD-40 repeat family protein. 018771 980 19FW 6914 039074 270 20FW 8114 7RV -1223 1FW 3114 4FW 583 7RV -4700 10RV -4223 19RV -1523 19RV -423 28FW 3014 13FW 7514 AT5G67300 AT5G67290 AT5G67320 5’ FW RV 3’ -5874 -5234 -4268 +1 +89 1026 +1217 +3133 +7090 -5603 -5116 -4175 +1898 +2904 +3247 +2 +1292 +1986 +2920 5’ 3’ AT5G67310 AT5G67310 Cytochrome P450 family protein. 17&24FW 2914 062181 1247 083728 1348

  6. SALK Genotyping 018771 G.S. FW/RV primers Sample #1-9 Sample #10-19 Sample #20-29 Sample #30-36 Tubulin control G.S. FW/LB (LBb1) primers

  7. SALK result W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W 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 15 17 M Mutation homozygous Wild/Mutation heterozygous Wildtype homozygous WM W Checked 36samples of SALK #018771 but…… NO tDNA insertion within AT5G67300 in line#018771 Brandon, call SALK!!!

  8. How is AT5G67300 expressed in SRB? <RT-PCR> mRNA exists in all stages 14-DAP-embryo 30-DAP axis Leaf Flower Leaf Stem Flower Unf.-ovule Control AT5G67300 <Gene Chip data> Expressed highly in leaves. Higher in mature seeds.

  9. Arabidopsis Thaliana vs. others 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 +1 +88 +1005 +1217 U T R U T R Solanum tuberosum (Potato) 364a.a. 47% Identity, 56% Positive similarity tuber-specific and sucrose-responsive element binding factor 173-249 87% 312-408 81% Nicotiana Tabacum (tobacco) 329a.a. 62% Identity, 73%Positive similarity Myb-like protein

  10. WHAT is AT5G67300? 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? 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

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