Partial functional characterization of the promoters CP1+ and CP2-, derived from a cassava
Glutamic Acid-rich protein gene, in cassava and carrot transgenic plants
Beltrán J1, Ladino J1, Vacca O1, López D1, Al-Babili S2, Beyer P2, Chavarriaga P1 and Tohme J1
1. Conservation and Use of Tropical Genetic Resources, CIAT, AA 6713 , Cali, Colombia 2. Albert-Ludwigs-Universitat Freiburg, Center for Applied
Biosciences, Schanzlestr 1, D-79104, Freiburg, Germany
INTRODUCTION • GUS expression localization
• To obtain a Golden Cassava trough genetic engineering it is Cassava: Although GUS expression was detected in leaves and fibrous
recommended to isolate promoters that drive the expression of roots, strong GUS staining was also seen in storage-roots. In a close up
carotenogenic genes in the storage root it is more noticeable the even distribution of the staining, which covers
cambium, secondary xylem and parenchyma cells. The strongest
• A Glutamic Acid-Rich Protein (GARP) was shown to be expression is seen in the cambium zone, which may also include phloem
preferentially expressed in cassava roots (De Souza et al 2002). Our cells.
goal was to evaluate two new versions of promoters isolated from
GARP, CP2- and CP1+, in cassava and carrot transgenic plants. CP2-
D a
is a shorter version of the promoter, without the first intron of the
b
gene, while CP1+ is a longer version with the intron.
METHODS
d
We transformed cassava’s friable embryogenic callus (FEC) of clone c
60444, and cotyledons and hypocotyls of carrot with A. tumefaciens
starin Agl1.
A: Storage roots of control (NT) and transgenics (17, 22); B
2x 35S hpt II CP version Gusplus and C: Stained slices of storgae roots from transgenic and
LB RB non-transgenic, respectively; D: close-up of stained root. a.
Cortical Parenchyma and Phloem. b. Cambium c. Fully
Plasmid containing either of two versions of a putative root- developed secondary xylem parenchyma cells. d. Primary
specific promoter (CP1+ or CP2-) driving the expression of xylem.
GUSplus.
Carrot: The expression driven by CP1+ in leaves was limited to the
vascular tissues of the central and lateral veins. In petioles, the
expression was confined to vascular bundles, while in roots the activity
was strong in the parenchyma and cambium cells.
A B C
Carrot and Cassava transgenic lines Leaf Petiole Mature root
Close-up of CP1+ activity in a carrot leaf (A), vascular
RESULTS AND DISCUSSION bundles of petiole (B) and storage root (C).
• Real-time PCR transgen detection
Cassava Carrot:
Transgenes Gus plus and hptII were detected in at least 11 cassava
•Expression was •Expression mainly
transgenic lines
widely distributed, in in the vascular
1kb 5 6 10 13 17 22 24 25 26 33 34 NT H2O P vascular and tissues
GUSplus parenchyma tissues
hpt II Control (A), transgenic Control (D), transgenic
leaves (B and C) leaves (E and F)
• Quantifying GUS mRNA levels
GUSplus expression in cassava: Leaf Vs Root
Several cassavas lines were transferred to field (May 2008) for final
2
expression analysis in mature, field-grown plants.
Relative mRNA levels (GUSplus )
1.8
1.6
1.4
1.2
1
0.8
Leaf
Root CONCLUSIONS AND PERSPECTIVES
0.6
0.4
0.2 • The CP1+ promoter from cassava seemed to have strong expression
0
22 17 in secondary phloem cells of carrot roots, indicating a potential to
Cassava transgenic lines
direct the expression of genes of interest in this root.
The results suggest that CP2- is highly expressed in both tissues, at • Although CP2+ activity was not exclusive for cassava storage root, it
least in the two lines analyzed here, indicating that the promoter may had a strong activity in root parenchyma cells , were the starch
not be that specific for storage roots. accumulation occurs.
Date prepared: July 2008