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THE FUNCTION OF AT5G04410 ( NAC2 ) AND AT3G10500 ( NAC2-like ) NAC Family

THE FUNCTION OF AT5G04410 ( NAC2 ) AND AT3G10500 ( NAC2-like ) NAC Family. YUYA HIRAI. A Cartoon Structure of My First Gene (At5g04410). 5 ’. 3 ’. 1. 3257. intron. exon. 3’UTR. 5’UTR. 6 exons and 5 introns 3257bp Forward orientation No apical meristem(NAM) family protein 567aa.

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THE FUNCTION OF AT5G04410 ( NAC2 ) AND AT3G10500 ( NAC2-like ) NAC Family

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  1. THE FUNCTION OFAT5G04410 (NAC2)ANDAT3G10500 (NAC2-like)NAC Family YUYA HIRAI

  2. A Cartoon Structure of My First Gene(At5g04410) 5’ 3’ 1 3257 intron exon 3’UTR 5’UTR • 6 exons and 5 introns • 3257bp • Forward orientation • No apical meristem(NAM) family protein • 567aa

  3. In What Organs Is the At5g04410 Gene Being Transcribed? Confirm the expected size band WT silique +RT WT silique -RT WT leaf -RT WT leaf +RT Water (neg.) gDNA (pos.) This gene is being transcribed in both leaf and silique. WT Leaf +RT Is this gene important for the seed development? Tubulin (440bp) 280bp 200bp To answer this question, search for plants having T-DNA inserted to this gene. The picture of RT-PCR

  4. The Location Site of T-DNA Fw primer -100 5’ 3’ 1 3’UTR 3257 583 intron exon 5’UTR 3100 T-DNA Rv primer LBb1 SALK_025098 Reverse orientation Location site 583bp Wild-Type band - 3.2 kbp T-DNA band - 0.8 kbp

  5. Which Plant Have T-DNA Inserted In the At4g04410 Gene? Isolation of gDNA from 14 plants (Fig.1) Fig.1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Fig.2 gDNA RNA Fig.2 Fig.4 Genotyping(Fig.2) Wild type: 7 plants Homo: 3 plants Hetero: 4 plants WT(3.2kbp) T-DNA(0.8kbp) The size of T-DNA band is about 0.8kbp Expected result! Expected result! Excise the marked band and sequence

  6. Nomarski Images (At5g04410) Wild type Walking stick Walking stick Homozygous mutant globular Torpedo There is no difference in phenotypes between wild type and mutant!

  7. A Cartoon Structure of My Second Gene (At3g10500) 3’ 5’’ intron 1 2419 exon 3’UTR 5’UTR • 6 exons and 5 introns • 2419bp • Forward orientation • No apical meristem(NAM) family protein • 549aa

  8. In What Organs Is the At3g10500 Gene Being Transcribed? Confirm the expected size band WT leaf +RT Leaf (from TA) gDNA(pos.) gDNA(pos.) WT silique -RT Seed(from TA) WT silique -RT WT silique +RT Water(neg.) WT silique +RT Water(neg.) WT leaf -RT WT leaf -RT WT leaf +RT This gene also is being transcribed in both leaf and silique! 0.4kbp Expected size -Tubulin +Tubulin Is this gene important for the seed development? To answer this question, Search for plants having T-DNA inserted to this gene

  9. The Location Site of T-DNA Fw primer 3’ 5’’ 38 3’UTR 2189 intron 1 2419 exon 5’UTR 2407 Rv primer T-DNA LBb1 SALK_018311 Reverse orientation Location site 2189bp Wild-Type band - 2.4kbp T-DNA band - 2.2kbp

  10. Which Plants Have T-DNA Inserted In the At3g10500 Gene? Isolation of gDNA from 14 plants (Fig.1) Fig.1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 gDNA RNA Fig.2 Pos. Neg. 1 2 3 4 5 6 7 8 8 9 10 11 12 13 14 Genotyping (Fig.2) Wild type: 1 plant Homo: 7 plants Hetero: 6 plants WT(2.3kbp) T-DNA(0.3kbp) The size of T-DNA band is about 0.3kbp Not expected result! Excise the marked band and sequence Very interesting result!

  11. The Location Site of T-DNA From My Experiment Fw 3’ 5’’ 38 3’UTR intron 2189 1 2419 exon 5’UTR 2407 Rv T-DNA LBb1 LBb1 SALK_018311 Reverse orientation Location site 2189bp Forward orientation

  12. Nomarski Images (At3g10500) Wild type globular torpedo Homozygous mutant globular torpedo There was no differences in phenotypes between WT and mutant plants!

  13. The Suggestion of My Experiment There 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!

  14. Thanks everyone especially…Dr Goldberg and Kunio!!! (of course Tomo!!) yu_yan_yu_yan_yu_yan@yahoo.co.jp

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