Transcript Identification and Molecular Characterization of Circadian Clock

By Matthew Geniza
Mentors: Dr. Todd Mockler and Dr. Sergei Filichkin
 To provide a foundation of the molecular
mechanisms that determine plant adaptations
depending on their environment.
 To increase the basic knowledge of how transcription
factors relate to phase-specific gene expression
depending on the external conditions.
 If Myb-10 is a clock gene, over expressing or
suppressing it alters the expression of other clock or
clock-regulated genes.
The plant circadian clock - at least three
interlinked loops
PRR5
PRR7
PRR9
INPUT
CCA1
LHY
light
OUTPUT
flowering
germination
growth
temperature
X
Y
GI
photosynthesis
metabolism
TOC1
LUX
Plants integrate daily temperature and light information into the circadian clock in order to gauge the
time of day and season, and thus coordinate biological processes appropriately for the environment.
 Plants have evolved an endogenous circadian clock
 Internal biological processes are synchronized with
changes in the environment.
 RNA transcripts of diurnal and circadian regulated
genes peak at certain times in the day and night.
 Understanding the regulation of gene expression is
important in the event of major climate changes
 Rice is crop plant that
serves as a stable food
for a majority of the
world.
 Rice has a sequenced
and annotated genome
 Is Myb-10 a clock gene? Does it control oscillations
of mRNAs of other rice genes? Does it affect the
phases of other clock genes?
 Will phase shifting caused by Myb-10 mis-expression
changes in phenotype, i.e. growth rate, flowering
timing, and ultimately – seed production?
 If yes, then Myb-10 is a clock-associated regulator.
Normalized Expression
1.2
A. thaliana
Rice Myb10 GeneID: Os05g34110
Poplar
Putative Myb-like TF
AT5G05090
1
0.8
0.6
0.4
0.2
0
0
4
8
12
16
20
24
28
32
36
40
Phase
http://diurnal.cgrb.oregonstate.edu/
44
 If the Myb-10 gene is a core clock gene, then it will
change the oscillations of other core clock genes.
 If the Myb-10 gene is NOT a core clock gene, then it
will change oscillations of other diurnally regulated
genes, but not core clock genes.
 To identify diurnally regulated genes in rice that are
affected by over expression or silencing of a specific
circadian clock regulated transcription factor.
 To identify cis elements in the promoter region of
Myb-10 which regulate its diurnal oscillations.
 Rice plants are grown in 12 hours of light and 12




hours of dark cycle for 7 days. This is followed by 60
hours of continuous light.
Tissue samples from plants are collected every 4
hours during the continuous light period.
RNA isolation
Bioanalyzer
Assayed by RT PCR to determine cycling profiles of
diurnally regulated gene targets
 Microarray
 The cycling of transcripts of several clock genes
(such as CIRCADIAN CLOCK ASSOCIATED 1) is
studied in the wild type rice and transgenic plants
expressing or suppressing Myb-10.
RIN 10.0
RIN 7.0
 Gel Image
Indica LDHC CCA1
0 4 8 12 16 20 24 28 32 36 40 44 48
Diurnal LDHC CCA1
Indica LDHC PRR95
0 4 8 12 16 20 24 28 32 36 40 44 48
Diurnal LDHC PRR95
 Quantitative data
 Measures gene
expression
 Assays all genes
 Analyze Microarray Data for Indica
 Prepare transgenic Myb-10 plants for Microarray
 Analyze Microarray Data for Myb-10 plants
 Compare Indica and transgenic Myb-10 plants
 Dr. Todd Mockler
 Dr. Sergei Filichkin
 The Mockler Lab
 Dr. Kevin Ahern
 HHMI