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

In What Organs Is the At5g04410 Gene Being Transcribed? Confirm the expected size band WT silique +RT WT leaf -RT WT silique -RT WT leaf +RT gDNA (pos.) Water (neg.) 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

The Location Site of T-DNA Fw primer -100 5’ 3’ 1 583 3’UTR 3257 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

Which Plant Have T-DNA Inserted In the At4g04410 Gene? Fig.1 1 2 3 4 5 6 Fig.2 7 8 9 10 11 12 13 14 Isolation of gDNA from 14 plants (Fig.1) 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! Excise the marked band and sequence Expected result!

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

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

In What Organs Is the At3g10500 Gene Being Transcribed? Confirm the expected size band WT leaf +RT WT silique +RT WT silique -RT Leaf (from TA) Seed(from TA) gDNA(pos.) Water(neg.) WT silique +RT WT silique -RT gDNA(pos.) 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

The Location Site of T-DNA Fw primer 3’ 5’’ 38 3’UTR 2189 1 intron 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

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!

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

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

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!

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