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Transcript
NAC Family Genes AT1G01720 AT1G77450 Yosuke Ishida From Kyoto Japan Basic information on my genes Gene 1 AT1G01720 1.1kbp 1 142 302 388 666 754 1186 1490 1129 T-DNA ATAF1 coding gene. SALK_057168 Transcript level increases in response to wounding. Activate CaMV35S promoter in Saccharomycos. Gene 2 AT1G77450 1.2kbp 1 76 245 347 622 711 1029 1308 484 T-DNA SALK_087702 Function of protein is not known yet. Positive cont WT WT Determination of genotype and T-DNA site (gene1) Plant Number PN 1 2 3 4 5 6 7 8 9 10 11 12 Genotype Homozygous wild type Heterozygous mutant Homozygous wild type Homozygous wild type Homozygous mutant Homozygous wild type Homozygous wild type Heterozygous mutant Homozygous wild type ----Homozygous mutant Homozygous wild type 1613 1036 506 Gel purification Sequencing reaction using LBb1 primer T-DNA inserted site is same as that shown in web site 1 142 302 388 LBb1 1129 666 754 T-DNA 1186 1490 Negative cont Positive cont WT WT Determination of genotype and T-DNA site (gene2) Plant Number PN 1 2 3 4 5 6 7 8 9 10 Genotype Homozygous mutant Heterozygous mutant Homozygous wild type Heterozygous mutant Heterozygous mutant Heterozygous mutant Homozygous wild type Homozygous mutant Homozygous wild type Heterozygous mutant 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. 1 76 245 347 LBb1 T-DNA 374 622 711 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 In which organ or tissue my genes is expressing? Gene 2 Gene 1 AT1G77450 AT1G01720 0.2kbp Poly(A) mRNA 0.7kbp PCR RT cDNA 0.2kbp 0.2kbp bands are expected PCR mRNA PCR Poly(A) RT cDNA 0.6kbp PCR 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 1036 1036 506 Tublin Bands 506 396 Supporting genechip data Positive cont Seed+RT Leaf+RT Silique-RT Silique+RT Leaf-RT Leaf+RT Positive cont Negative cont Silique+RT Silique-RT Leaf -RT Leaf+RT Cloning promoter region undigested plasmid vector AT1G01720 2.4 kbp PCR 12216 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 1613 1.9 kbp EcoRI EcoRI 3.9kbp gDNA vector+inserted fragment Sequencing analysis to find out mutation AT1G01710 AT1G01720 AT1G77440 ---t-----a--- ---tag-----ttgtta--SP6 T7 SP6 T7 2.4 kbp 0.7kbp SP6 AT1G77450 0.8kbp T7 Mutation sequence locates to 5’UTR of gene 0.8kbp 1.9 kbp SP6 0.8kbp T7 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.
