Transcript Arabidopsis thaliana gene At3g50060

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