Promoter
Comparative functional identification and analysis of Carica papaya promoter in the model system Arabidopsis thaliana revealed post-transcriptional regulation of gene expression
INTRODUCTION
a
a
CaMV35S
Poly-A
hptII
b
5’
3’
RB
eGFP
Promoter
Construct
b
1975
66
759
42
759
Cp29-eGFP
1796
36
759
75
759
759
Primer
3’
5’
Transcribed mRNA chain
36
39
42
45
Cp45
WT
27 kDa
7 DAP
600bp
14 DAP
14 DAP
21 DAP
28 DAP
Ac n2
Ac n2
45 kDa
Ac n 2
100000000
10
20
30
40
50
Promoter Amplicon Size (bp)
5’-ctgcagtcttgaatcttgtaacgactagattcc-3’
5’-ccatggTCTCTGCTAAAGCGGAGAAAGCTGAGA-3’
GENE
Cp29F
5’-ctgcagggccatgacttcgagtcttcggttag-3’
Cp29R
5’-ccatggggccaaatatttgagagatgagaaacc-3’
5’-ctgcagctactgcctactttaccatgtggtag-3’
5’-ccatggggttactggttggtgggtgaatgaattg-3’
Cp45F
5’-ctgcagggacagtttgagtattacaacagaG -3’
Cp45R
5’-ccatggacaagttagtaaacaaacacaaatggctg-3’
RATIONALE
HASTEN the time to screen, identify and characterize promoters from papaya.
PAPAYA TREES
Long generation time
•
•
Genome size
(372 Mbp)
Isolate STRONG NATIVE PAPAYA PROMOTERS from genes UP-REGULATED by P. palmivora &
“switch on” in roots & stem (primary targeted tissues) HO
MW
Cp9
Cp29
Cp35
Short life cycle Cp 29 - Beta-1,3-glucanase
Cp47 (actin)
EtBr
Easy to transform 24 hour post inoculation of P. palmivora
MATERIALS & METHOD
•Identify root and stem expressed genes from papaya.
•Clone the 5’-upstream 2.0 kilobase regions containing putative promoters.
35 S
Small genome (135Mbp)
Cp9
•Assess promoter activity in Arabidopsis tissues:
• Visual, GFP fluorescence microscopy
• GFP RNA level ( RT-PCR )
• GFP Protein level ( immunoblot GFP antibody )
• 5’ RACE Analysis
WT 35S
Cp35
Cp45
WT
27 kDa
Leaf
Stem
Ac n 2
45 kDa
Cp 9
20
Cp 29
Cp 35
15
Cp 45
INTRODUCTION
a
a
CaMV35S
Poly-A
hptII
b
5’
3’
RB
eGFP
Promoter
Construct
b
1975
66
759
42
759
Cp29-eGFP
1796
36
759
75
759
759
Primer
3’
5’
Transcribed mRNA chain
36
39
42
45
Cp45
WT
27 kDa
7 DAP
600bp
14 DAP
14 DAP
21 DAP
28 DAP
Ac n2
Ac n2
45 kDa
Ac n 2
100000000
10
20
30
40
50
Promoter Amplicon Size (bp)
5’-ctgcagtcttgaatcttgtaacgactagattcc-3’
5’-ccatggTCTCTGCTAAAGCGGAGAAAGCTGAGA-3’
GENE
Cp29F
5’-ctgcagggccatgacttcgagtcttcggttag-3’
Cp29R
5’-ccatggggccaaatatttgagagatgagaaacc-3’
5’-ctgcagctactgcctactttaccatgtggtag-3’
5’-ccatggggttactggttggtgggtgaatgaattg-3’
Cp45F
5’-ctgcagggacagtttgagtattacaacagaG -3’
Cp45R
5’-ccatggacaagttagtaaacaaacacaaatggctg-3’
RATIONALE
HASTEN the time to screen, identify and characterize promoters from papaya.
PAPAYA TREES
Long generation time
•
•
Genome size
(372 Mbp)
Isolate STRONG NATIVE PAPAYA PROMOTERS from genes UP-REGULATED by P. palmivora &
“switch on” in roots & stem (primary targeted tissues) HO
MW
Cp9
Cp29
Cp35
Short life cycle Cp 29 - Beta-1,3-glucanase
Cp47 (actin)
EtBr
Easy to transform 24 hour post inoculation of P. palmivora
MATERIALS & METHOD
•Identify root and stem expressed genes from papaya.
•Clone the 5’-upstream 2.0 kilobase regions containing putative promoters.
35 S
Small genome (135Mbp)
Cp9
•Assess promoter activity in Arabidopsis tissues:
• Visual, GFP fluorescence microscopy
• GFP RNA level ( RT-PCR )
• GFP Protein level ( immunoblot GFP antibody )
• 5’ RACE Analysis
WT 35S
Cp35
Cp45
WT
27 kDa
Leaf
Stem
Ac n 2
45 kDa
Cp 9
20
Cp 29
Cp 35
15
Cp 45