NAC Family Genes AT1G01720 AT1G77450 Yosuke Ishida From Kyoto Japan

Basic information on my genes AT1G01720 AT1G77450 1.1kbp 1.2kbp 1 142 302 388 666 754 1186 1490 1 76 245 347 622 711 1029 1308 1129 484 T-DNA T-DNA ATAF1 coding gene. SALK_057168 Transcript level increases in response to wounding. Activate CaMV35S promoter in Saccharomycos. SALK_087702 Function of protein is not known yet.

Determination of genotype and T-DNA site (gene1) Positive cont WT WT Plant Number 1613 1036 506 Gel purification Sequencing reaction using LBb1 primer T-DNA inserted site is same as that shown in web site LBb1 1129 T-DNA 1 142 302 388 666 754 1186 1490

Determination of genotype and T-DNA site (gene2) Positive cont Negative cont WT WT Plant Number 1613 1036 506 Gel purification Sequencing reaction using LBb1 primer T-DNA site is 107 bases upstream from that shown in web site. Inserted to 2nd exon. LBb1 374 T-DNA 1 76 245 347 622 711 1029 1308

Observation under Normarski to find phenotype of homozygous mutant Homozygous mutant of 1st gene Homozygous mutant of 2nd gene No significant difference between wild type and homozygous mutant

RT-PCR analysis AT1G77450 AT1G01720 In which organ or tissue my genes is expressing? Gene 1 Gene 2 AT1G77450 AT1G01720 0.2kbp 0.7kbp PCR PCR mRNA Poly(A) mRNA Poly(A) RT RT cDNA cDNA 0.2kbp PCR 0.6kbp PCR 0.2kbp bands are expected 0.6kbp band and 0.7kbp bands are expected Tublin primers are added with RT-PCR primers in order to confirm that cDNA was synthesized

Supporting genechip data Silique+RT Silique-RT Leaf+RT Leaf-RT Seed+RT Leaf+RT Silique+RT Leaf+RT Leaf -RT Silique-RT Positive cont Negative cont Positive cont 1036 1036 506 506 Tublin Bands 396 Supporting genechip data

Cloning promoter region undigested plasmid vector AT1G01720 gDNA 12216 vector+inserted fragment 2.4 kbp PCR 4072 3054 2036 1613 1018 EcoRI 1.9kbp 506 0.5kbp EcoRI EcoRI 3.9kbp AT1G77450 1.9 kbp PCR No EcoRI site 4072 3054 2036 1.9 kbp 1613 EcoRI EcoRI 3.9kbp

Sequencing analysis to find out mutation AT1G01710 AT1G01720 AT1G77440 AT1G77450 ---t--- ---tag--- ---a--- ---ttgtta--- SP6 T7 SP6 T7 2.4 kbp 1.9 kbp 0.7kbp 0.8kbp 0.8kbp 0.8kbp SP6 T7 SP6 T7 Mutation sequence locates to 5’UTR of gene 255th ‘T’ downstream from 3’UTR end mutated into ‘A’ It is necessary to carry out sequencing reaction. It is possible that database is just differrent from my sequencing result.

Summary and future work Background 2 genes belong to ATAF subfamily of NAC family. They have very similar DNA sequence. Result of experiment Phenotype of homozygous mutant plants are wild type. Similar expression pattern (genechip) My hypothesis is one gene complement another If one gene lost function, another works instead of it. Expression study of KO plant would reveal the relationship between 2 genes.