Transcript HC70AL Final Presentation

HC70AL
Final Presentation
Chris McQuilkin
June 4th, 2009
Gene One: AT4G36540
• A transcription factor involved in DNA
binding
• A bHLH Gene
– “basic Helix-Loop-Helix” refers to the
structure of the protein
– bHLH genes are found in Eukaryotes and
are highly conserved across species
AT4G36540
What is the Structure of the Gene?
T-DNA Insertion
LB
Site
Translational
Start Codon
Intron 1
116
Translational
Stop Codon
Forward
Primer
288
Exon 1
85
Intron 2
180
Exon 2
Total length= 1,714 base
pairs
92
Intron 3
207
Exon 3
91
Reverse Primer

Intron 4
134
Exon 4
91
93
Exon 5
331
Where is the Gene Active?
CM Arabidopsis RT-PCR Gene:
AT4G36540 (Leaf and Silique cDNA)
GDNA
ScDNA
LcDNA
100 bp
•Why is there no band in the positive control?
RT-PCR Forward Primer
Intron
Forward Primer
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mRNA Accumulation
Where is the Gene Active?
mRNA accumulation
2500
2000
1500
Series1
1000
500
0
Plant region/ stage of development
Where is the Gene Active?
Promoter Cloning
Strategy of Promoter Activity
Analysis
Arabidopsis Genomic DNA
•PCR amplification of upstream region
•With Gene-specific Primers
•And High Fidelity DNA Polymerase
PCR Product
pENTR/D-TOPO vector
Ligation : Population of Recombinant Plasmid
(vector+PCR product) and NON-recombinant plasmid
(vectory only)
Transformation of competent E.coli cells
Screening for E.coli cells harboring recombinant
plasmid
Confirmed Recombinant plasmid DNA: Verifying the authenticity of
recombinant plasmid DNA by Restriction Enzyme Digestion
DNA sequences : verification of the cloned
Promoter Region by Sequencing Analysis.
Sequence Analysis and confirmed identity of the
cloned upstream region
Recombinant Plasmid DNA + BetaGluronidase (GUS) gene carrying T-DNA
Vector
Krista Templeton and Auni
Hovanesian, HC70AL 2008
Promoter Cloning
PCR of AT4G3540
Promoter Region
1 hr 120 volts
*Two fragments were
amplified by PCR
~1.7 kb= Expected Size of PCRamplified promoter
~0.8 kb= Unexpected PCR product
Positive Control
iProof Polymerase
1 kb Ladder
Promoter Cloning
Expected Plasmid alone=
2.5 kb
AscI-Digested pENTR
Plasmid DNA From
Six E. coli Colonies
Expected Plasmid +
Promoter= 4.2kb
Observed= 2.5kb and
3.5 kb
Gene one: Genotyping
1 hr, 120 volts
Wild-type
Control
Gene one: Genotyping
04/16/2009
1 hr, 120 volts
Why are there
two bands in the
Lanes 1 and 3?
~1000 base pairs
~900 base pairs
~250 base pairs
Hemizygous
Hemizygous
~250 bases
Homozygous
Hemizygous
What is the expected size
of the T-DNA band?
What is the expected size
of the Wild-Type Band?
887 Bases
Concatamers
T-DNA
Insertion Site
LB 
LB
Reverse Primer
Translational
Start Codon
Forward
Primer
Intron 1
119
116
288
Exon 1
85
Intron 2
180
Exon 2
92
Translational
Start Codon
Intron 3
207
Exon 3
91

Intron 4
134
Exon 4
91
93
Exon 5
334
331
Nomarski Observation
Mutant Embryo and wild-type embryo show no phenotypic differences
Nomarski Observation
Mutant seed coat and wild- type seed coat show no phenotypic
differences
Results
• Three hemizygous and one homozygous
T-DNA plants were identified
– Two bands were observed in each of the
lanes containing DNA with a T-DNA insert
• Gene AT4G36540 mRNA accumulation
was observed in both the leaf and silique
of Arabidopsis
Results
• The promoter did not insert into any of the
E. coli plasmids that were screened.
– Four of the colonies contained an unidentified
insert about 1 kb long
• No phenotypic changes were observed in
either the hemizygous or homozygous TDNA Arabidopsis plants using Nomarski
Observation
Conclusion
• AT4G36540 is not lethal, or there may be
another DNA sequence that codes for the
same protein
• Although no phenotypic differences were
observed in the seed coat or embryo using
Nomarski, there could be differences that
are more subtle or appear at different
stages of development
What next?
• Look further for phenotypic changes in Arabidopsis
mutants—examine different tissues and more stages of
development
• Sequence the unidentified DNA fragment taken up by the
E. coli C600 plasmids
• Repeat PCR of the promoter
– Excise the band containing recombined plasmid DNA, and
repeat transformation
– If a promoter is identified, use GUS to study where the gene is
expressed
• Grow more plants from the identified mutant lines
• Repeat sequencing reaction of region between LB and
T-DNA primers to confirm location of the T-DNA insert
Gene Two: AT1G75240
• A homeobox gene
• Homeobox genes regulate development
and cell differentiation
– A homeobox is a sequence
• They are found in animals, plants, and
fungi
AT1G75240
What Is The Structure of the Gene?
Predicted T-DNA Insert
LB
Reverse Primer
Forward Primer
330
77
97
927
Translational Start
Codon
Total Length: 1,685 Base Pairs
254
Translational Stop
Codon
Where is the Gene Active?
AT1G75240 mRNA in
Arabidopsis Leaf and Silique
1 hr 120 volts
Leaf cDNA
Silique cDNA
Tubulin mRNA
AT1G75240
mRNA
Gene is active in the silique but not the
leaf
3500
3000
2500
2000
1500
1000
500
0
Developmental Stages
Seed/Post-Mature
Green Stage (18-
Seed/Mature Green
Stage
Seed/Cotyledon
Stage (7-8DAP)
Ovule/Reproductive
Embryo
Proper/Mature
Embryo
Proper/Heart Stage
Average mRNA Accumulation
Where is the Gene Active?
mRNA Accumulation of AT1G75240
Series1
Genotyping
100 bp
LBb1 Control
WT Control
Arabidopsis 12
Arabidopsis 11
Arabidopsis 10
Arabidopsis 9
Arabidopsis 8
Arabidopsis 7
Arabidopsis 6
Arabidopsis 5
Arabidopsis 4
Arabidopsis 3
Arabidopsis 2
Arabidopsis 1
100 bp
Results
• All the plants screened were wild-type
Conclusion
• The chance of getting all wild-type plants
is extremely low, so it is possible that the
Salk Institute sent the wrong kind of plant
Thank you!
•
•
•
•
•
•
•
Anhthu Bui
Brandon Chen
Bob Goldberg
Daisy Robinton
Ingrid Nelson
Kristin Gill
Min Chen
SRB Contig Sequencing
Contig Length: 50.7 KB
Approach
• Contig entered into three online databases that look
for predicted genes: FGENESH, GENSCAN, and
GeneMark
2,000
1
240
396 - 507
1134 - 1442
628- 729 759-846 999-1097
396-507
1134-1642
1324-1428
999-1092
1901 -
18431901-
459- 572
281- 427
2,001
3272
-2550
-2550
-2086
2126-2351
2990
2894 3046
4,000
Approach
• Predicted Genes were entered into a
BLAST search to see if the predicted gene
matched Expressed Sequence Tags
(ESTs) found in other species
Approach
• Whole Contig was searched for DNA
repeats
Results: GENSCAN
Accession Number
E-value
Description
1
BP033723.1
1e-110
Lotus Japonicus
2
FN014331.1
1e-52
Petunia axillaris subsp.
axillaris pool of root and
petal tissue
3
GO259467
3e-26
Tissue from peanut Arachis
hypogaea
4
EX522455.1
0.0
Trichome from stem of
Medicago sativa
5
FK023593.1
8e-77
cDNA from Glycine max
(soy bean)
Predicted Gene (BLASTN)
Results: GENSCAN
Predicted Protein (tBLASTn)
Accession Number
E-value
Description
1
Y18169
5e-70
Pisum sativum mitochondrial
ccb248 gene and partial rps7
gene.
2
FM179380
2e-10
Vitis vinifera complete
mitochondrial genome,
cultivar Pinot
noir clone ENTAV115
3
L40816
3e-35
4
DQ008791
5e-60
Thottea tomentosa large
subunit ribosomal RNA gene,
partial sequence;
mitochondrial
5
AC144406
4e-14
Medicago truncatula clone
mth2-5h18, complete
sequence.
2e-41
hypothetical protein
NitaMp002 [Nicotiana
tabacum].
Predicted Protein (BLASTp)
4
5
YP_173352
No significant match
Results: FGENESH
Accession
Number
E-value
Description
1
EX527626
0.0
MTGland_A031_2007-05-22/MTGlandA031_B09_039_1 Medicago truncatula
A17 glandular trichome Medicago truncatula cDNA, mRNA sequence
2
CD721224
2e-25
Chardonnay Vitis vinifera
3
GD544655
1e-40
Scarlet Runner Bean globular-stage suspensor region
5
FN014331
9e-88
Pool of root and petal tissue of Petunia axillaris
6
FF554629.1
9e-143
Vigna Unguiculata
1
Y18169
18e-118
Pisum sativum mitochondrial ccb248 gene and partial rps7 gene.
6
AC144406
8e-18
Medicago truncatula clone mth2-5h18, complete sequence
CAB43024
2e-118
cytochrome c biogenesis protein [Pisum sativum]
Predicted
Gene
(BLASTn)
4
Predicted
Protein
(tBLASTn)
Predicted
Protein
(BLASTp)
1
Results: GeneMark
Predicted Protein
(tBLASTn)
Accession Number (of Lowest
E-Value)
E-value
Description
1
AC192958
1e-13
Medicago truncatula
chromosome 2 BAC
clone mte1-45m19,
complete
sequence
2
AC147537
1e-23
Medicago truncatula
clone mth2-133k2,
complete sequence.
3
EX527626
3e-113
Glandular trichome
Medicago truncatula
13
BA000042
1e-18
Nicotiana tabacum mitochondrial
DNA, complete
genomeLength=430597
15
L40816
5e-25
Glycine max
mitochondrion
polymorphic marker
DNA sequence.
16
L40816
7e-46
Glycine max
mitochondrion
polymorphic marker
DNA sequence.
17
AC145156
1e-50
Medicago truncatula
clone mth2-7h6,
complete sequence.
18
DQ647831
1e-108
Chlorokybus atmophyticus
large subunit
ribosomal RNA gene,
partial
sequence;
mitochondrial.
Results: Gene Mark
19
BA000042
1e-41
Nicotiana tabacum
mitochondrial DNA,
complete genome.
20
AP004975
1e-26
Lotus japonicus
genomic DNA,
chromosome 5, clone:
LjT21J12, TM0158,
complete
sequence.
21
AP004975
7e-17
Lotus japonicus
genomic DNA,
chromosome 5, clone:
LjT21J12, TM0158,
complete
sequence.
23
XM_002336124
2e-12
Populus trichocarpa
predicted protein,
mRNA.
32
FM179380
5e-29
Vitis vinifera
complete
mitochondrial genome,
cultivar Pinot noir
clone
ENTAV115.
33
AC144406
7e-29
Medicago truncatula
clone mth2-5h18,
complete sequence.