Download Molecular Biology of the Peribacteroid Membrane

A reverse-genetic screen for
N-regulators
•
UNDERLYING ASSUMPTIONS
– Transcription factors (TFs) are involved in N-regulation
– Some of these TFs are regulated at the transcriptional level by
N availability
•
SCREENING STRATEGY
– Identify N-regulated TF genes by real-time RT-PCR
– Over-express suspect TF genes in Arabidopsis (using
constitutive and inducible promoters)
– Obtain TF knockout mutants for genes of interest
– Determine the phenotype of mutant and transgenic lines
– Test expression of N-assimilation genes in mutants and
transgenic lines
– Confirm physical interaction between TF and promoters (e.g.
ChIP-PCR)
Real-time RT-PCR profiling of >1400
Arabidopsis transcription factor genes
90% coverage with high specificity, sensitivity, dynamic range,
and robustness
45
A
CT Value
40
35
30
25
2
R =0.999
20
2
R =0.997
15
100
101
102
103
104
105
106
Double-stranded Template Copy Number
Fraction of Shoot cDNA
1.0
Expression Level (2
(40-CT)
)
125000
0.8
0.6
0.4
0.2
0.0
B
2
100000
R =0.996
75000
2
R =0.998
50000
2
R =0.993
25000
2
R =0.994
0
0.0
0.2
0.4
0.6
Fraction of Root cDNA
Czechowski et al. (2004) Plant J. 38, 366-379.
0.8
1.0
Real-time RT-PCR is more
precise than Affy chips
Real-time RT-PCR
Intra-assay (A) and
Interassay (B) variation
Affy Chips
Interassay variation
N-regulated TF genes: Suspects for
global control of N acquisition
Axenic culture: +N to –N to NO3-
Log2 TF ratio X/+N
10
6
2
0
-2
-6
-10
Inducible over-expression
to identify target
genes/phenotypes
+N
-N2d
NO3-30min
40 N-regulated TFs, including
regulators of anthacyanin
biosynthesis and flowering
(FD).
Scheible et al. (2004) Plant Physiol. (in press).
The role of transcription
factors in abiotic stress
tolerance and
plant development
Overview of TF projects
-N
NO3-
-P
Redox
Real-time RT-PCR/
Reverse genetics
-S
Heterosis
Salt+H2O
stress
Seed
Develop.
Seed/silique-specific TFs: potential
tools for seed biotechnology
Shoots (log2)
TF gene expression level relative to ubi10
50-fold induced: candidates
Siliques (log2)