Arabidopsis thaliana gene At3g50060 - PowerPoint PPT Presentation

Title: Arabidopsis thaliana gene At3g50060


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Arabidopsis thaliana gene At3g50060

• A Gene Story
• HC70AL
• Spring 2004
• Gloria Chu

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The Gene
Gene Number At3g50060 Gene Name AtMYB77 or
AtMYBr2 Function myb transcription factor
5 1
3 1135
UTR
Gene AT3G50060
Exon
The Protein
Scarlet Runner Bean EST

• Codes for 301 Amino Acids
• Similar protein found in Solanum tuberosum

• 508 bps
• Aligns with At3g50060 for approximately 216 bps
• - 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/whatsmyb2
0short.htm
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Gene Activity
Odd Lanes are RT Even Lanes are -RT controls
Control Band
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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?
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Madison Screen Results
Round 1 Superpools
Round 2 DNA Pools

• DNA Pools from Superpool 22
• 2nd DNA Pool (Pool 191) shows clear band at
  approximately correct size of At3g50060 fragment
  (1496 bps)

Superpools amplified using gene specific FW primer
Superpool 22
Superpool 2
L 1 2 3 4 5 6 7 8 9 C L
Superpools amplified using gene specific RV primer
JL202 Primer
22 Fw
Gene-specific Fw primer
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SALK InsertIons
HITS SALK_055373.34.15.x TYPE SALK T-DNA TDNA
EVAL 5e-88 LOCN 300-UTR5 COOR
C/18569907-18570052
HITS SALK_067655.56.00.x TYPE SALK T-DNA TDNA
EVAL e-139 LOCN Exon COOR C/18569060-18569313
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Graphical Depiction of Gene At3g50060 and T-DNA
Insertions
055373
067655
22 FW
2FW
5
3
FW
RV
Primers
Intron
Exon
SALK
Madison
Locations of T-DNA Insertions Madison
SALK
Superpool 2Fw 1117 (3UTR)
Line 067655 776 (exon) Superpool 22Fw 965
(3UTR) Line 055373 37 (5
UTR)
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SALK Plant Genotypes
L 1 2 3 4 5 6 7 8 9 WT Neg. Control
Gel 1 Wild-Type Allele Gene-specific Fw and Rv
primers Gel 2 Mutant Allele Gene-specific Rv
primer and LBb1
L 1 2 3 4 5 6 7 8 9 WT N P
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SALK Plant Phenotypes
Wild Type Plant (homozygous WT)
COMING SOONDue to technical difficulties
Mutant (Knockout) Plant (homozygous mutant)
COMING SOONDue to technical difficulties
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Back to the Big Questionand 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