Download Work_presentation_Mar1808

Work presentation
Gaurav Moghe
Feb 4th , 2008 – March 17th, 2008
Projects
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Divergence between DDF1 and DDF2
Expression differences between A.thaliana
and A.lyrata under JA-induced stress
Novel coding regions in A. thaliana
Amanitus-related toxins in other fungal
genomes
1. Divergence between DDF1 and DDF2
Objective:
To find protein-level differences between
DDF1 and DDF2 using translational fusions
Subobjective:
 Verify transformants for GUS marker
-- GUS Staining
-- Homo/Heterozygosity using GUS-specific primers
Strategy: Translational fusions
Endogenous
promoter
Endogenous
promoter
DDF1
DDF2
GUS
GUS
10 lines
1 line
Spatial and temporal differentiation of expression in Arabidopsis
But,
• Existing lines from older A. thaliana strains.
• Moving everything to the newer Col0 strains.
GUS Staining results
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Tried GUS staining from leaves, flowers,
roots and first inter-node
No GUS activity detected
Progeny of transformants not differentiable
by Hyg resistance phenotype
Future plans
1) New Col0 strains (DDF1-GUS) put on soil
2) Perform GUS staining with new Col0 strains
under salt-stress from roots and inter-node
3) Verify homo / heterozygosity using GUSspecific primers
ChIP-Sequencing part
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35S-cMyc-DDF1/2 construct (1 probable
plant)
35S-DDF1/2-GFP construct
Transformant should show over-expression
phenotype, but that was not observed for
GFP construct. So, will have to be re-done.
2. JA-induced stress expression
JA treated
Control
A. thaliana
2
2
A. lyrata
2
2
Extract RNA using RNAeasy
Solexa sequencing
qRT-PCR
Analysis
RNAEasy (Qiagen)
Sample
AT JA
AT JA
AT Control
AT Control
AL JA
AL JA
AL Control
AL Control
Yield
260/280
260/230
From 200Above 2.0 (low/none
600 ng/micL protein contamination)
DNA contamination
The extracted RNA samples (200ng) were
PCR-amplified using GoTaq with:
1) actin_6 (flanks an intron)
2) DDF2 primers
for 40X and 32X cycles.
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Both the reactions showed significant
presence of DNA (~20ng after amplification)
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DNA contamination: options
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The SuperArray RT2 First strand kit
(Problem: Two-step RTPCR. No kit mentioned for Eppendorf Realplex
machine)
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Design primers flanking the longest introns
If possible, keep a smaller extension time(?)
(Problem: Can affect RNA amplification)
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Keep RT(-) control during qRT-PCR
(Problem: What is the best way to subtract effect of DNA?)
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Re-do the extraction
3. Novel coding regions in A.thaliana
Two directions of study
Gene predictors
RNA genes
1. Augustus
2. GlimmerM
3. GeneID
4. TwinScan
BioPerl installation issue
5. EuGene
For some reason, not accepting Chr4
and Chr 5
6. CONTRAST
7. SNAP
Whole genome alignments
Predicting existence of yet un-annotated
RNA genes
Question:
Are there any regions in the genome that:
1) Are not known proteins/RNA?
2) Conserved across phylogeny?
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Are such regions:
1) RNA?
2) Protein?
RNA genes: Strategy
Download A.thaliana PUTs from PlantGDB
(best alternative to FLcDNA)
BLASTx against UniProt
(1)
RFAM database of
known/predicted RNA
molecules
(UniRef90)
Filter (1) using (2)
distribution
BLASTx against UniProt
(UniRef90) (2)
Retain non-matching
and not significantly
matching PUTs
Discard significant
matches
PUTs not known to be
proteins
RNA genes: Strategy
PUTs not known to be proteins
10000 randomly generated
PUTs for pre-selected size
classes of the original PUTs
BLASTn against RFAM
(1)
Filter (1) using (2)
distribution
BLASTn against RFAM
(2)
Retain non-matching
and not significantly
matching PUTs
Discard significant
matches
PUTs not known to be
proteins or RNA
RNA genes: Strategy
PUTs not known to be
proteins or RNA
Are these PUTs mapped to A.
thaliana genome?
Yes
BLAST against PUTs from
different plants strategically
placed in phylogeny
Conserved regions not yet
annotated
RNA prediction softwares
Find conserved PUTs
1)
RNAZ
2)
QRNA
3)
ddbRNA
4)
EvoFold
5)
MSARI
4. Amanita toxin project (Walton lab)
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Multiple sequence alignment of several toxin
genes from two fungal genera – Amanita and
Galerina
Sequenced fungal genomes
tBLASTn with relaxed parameters does not
give any significant hits
Use of Hidden Markov Model (HMM) to
generate a PWM for MSA of toxins
Ama-like
toxins MSA
M
ARLP
PC
E
HMM
HMM
Scan 6-frame
translated fungal
genomes
Scan 6-frame
translated fungal
genomes
Hits of very low e-value (100-1000),
but some degree of relevant sequence
similarity
Thank you