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Arabidopsis thaliana gene At3g50060 A Gene Story HC70AL Spring 2004 Gloria Chu Gene Number: At3g50060 The Gene 5’ Gene Name: AtMYB77 or AtMYBr2 Function: myb transcription factor 1 1135 3’ UTR Gene AT3G50060 Scarlet Runner Bean EST Exon The Protein •508 bps •Codes for 301 Amino Acids •Aligns with At3g50060 for approximately 216 bps •Similar protein found in Solanum tuberosum - from +152 to +367 of gene •From suspensor of SRB •PCSC11470: similar to At4g37260 (AtMYB73) “The hallmark of these proteins is a highly conserved DNA-binding domain composed of tandem repeats of a helix-turn-helix motif (light blue) that fit into the major groove of the DNA double helix (red and yellow).”* * http://www.stanford.edu/group/lipsick/whatsmyb%20short.htm Gene Activity Genechip Signal Detection for At3g50060 Av erage Signal Detection 2700 2400 2100 Level of Activity 1800 1500 1200 900 600 300 0 A verage Signal Detectio n Wild Type Ovules Wild Type 24-Hr Seeds Wild Type M idM aturatio n Seeds Wild Type M ature Green Seeds Wild Type P o stM aturatio n Green Seeds Wild Type Seedlings Wild Type Leaves Wild Type Ro o ts Wild Type Stem Wild Type C24 Leaves 831.75 847.25 1385.5 1828 2308.9 494.85 1562.3 391.9 1969.9 329.95 Stages of Plant Development Control Band Odd Lanes are +RT Even Lanes are -RT controls The Big Question Is my gene vital for seed development in Arabidopsis plants? The Big Question Reworded for the Sake of Experimentation Does knocking out my gene cause an embryo lethal mutation? Madison Screen Results Round 1: Superpools Superpool 22 Round 2: DNA Pools Superpools amplified using gene specific FW primer Superpool 2 DNA Pools from Superpool 22 •2nd DNA Pool (Pool 191) shows clear band at approximately correct size of At3g50060 fragment (1496 bps) L 1 2 3 4 5 6 7 8 9 C L Superpools amplified using gene specific RV primer JL202 Primer Gene-specific Fw primer HITS SALK_067655.56.00.x HITS SALK_055373.34.15.x TYPE SALK T-DNA [TDNA] TYPE SALK T-DNA [TDNA] EVAL e-139 EVAL 5e-88 LOCN Exon LOCN 300-UTR5 COOR C/18569060-18569313 COOR C/18569907-18570052 Graphical Depiction of Gene At3g50060 and T-DNA Insertions 055373 22 FW 067655 5’ 3’ FW Primers 2FW RV Exon Intron SALK Madison Locations of T-DNA Insertions Madison SALK Superpool 2Fw: +1117 (3’UTR) Line #067655: +776 (exon) Superpool 22Fw: +965 (3’UTR) Line #055373: +37 (5’ UTR) SALK Plant Genotypes L 1 2 3 4 5 6 7 8 9 WT Neg. Control Gel 1: Wild-Type Allele Genespecific Fw and Rv primers PLANT GENOTYPE 1 Homozygous wild-type 2 Homozygous wild-type 3 Homozygous mutant 4 Heterozygous mutant 5 Homozygous mutant 6 Homozygous wild-type 7 Homozygous mutant 8 Homozygous mutant 9 Homozygous mutant L 1 2 3 4 5 6 7 8 9 WT N P Gel 2: Mutant Allele Genespecific Rv primer and LBb1 SALK Plant Phenotypes Wild Type Plant (homozygous WT) COMING SOON… Due to technical difficulties Mutant (Knockout) Plant (homozygous mutant) COMING SOON… Due to technical difficulties Back to the Big Question and Where To Go From Here Knocking out gene At3g50060 does not cause an embryo lethal mutation Photo: A Greenhouse; not the UCLA greenhouse; just a greenhouse