Promoter

Topics: DNA, Gene expression, Gene Pages: 1 (828 words) Published: October 14, 2014
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...
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