Biosynthesis of Very Long Chain Polyunsaturated Fatty Acids in Chicory
Hattem Mekky1, Michael R. Davey1, J. Brian Power1 , Maged E. Mohamed2 and Colin M. Lazarus2
1Plant
and Crop Sciences Division, School of Biosciences, University of Nottingham, 2School of Biological Sciences, University of Bristol
Introduction:
Very long chain polyunsaturated fatty acids (VLCPUFAs) are declining in reserves, since they are produced by oily fish. VLCPUFAs are important in human nutrition because they are responsible for the
production of inflammatory mediators such as prostaglandins, the regulation of cholesterol synthesis and its transport, and the maintenance of cellular membranes. This research aims to produce
VLCPUFAs in edible plants.
Results:
3 4
5
6
7 8
500bp
175bp
2 3 4
5
1A
6
7
500bp
8
1B
Figure 1: Amplification of A,
the ∆9 elongase, B, the ∆8
desaturase and C, the ∆5
desaturase genes in leaf DNA
extracts.
Lane 1: positive control, lane
2: negative control. Lanes 3-8
(Figure 1A and C) and lanes 3,
6 and 8 (Figure 1B) are
representative examples of
transgenic plants.
Weight (g)
0.8
0.6
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0
5
10
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20
0
0
Conc. (mg/l)
40
60
80
100
Conc. (mg/l)
120
140
160
1.2
1.2
1
1
0.8
0.8
Weight (g)
Weight (g)
20
Effect of citric acid in presence of SM and RM
Effect of caffeic acid in presence of SM and RM
0.6
0.4
0.2
0.6
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20
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250
500
Conc. (mg/l)
750
1000
1250
1500
Conc. (mg/l)
Effect of Jasmonic acid in presence of SM and RM
Effect of nicotinic acid in presence of SM and RM
1.4
1.2
1.2
1
W eigh t (g)
1
Weight (g)
4. Cell suspensions were initiated from ∆9 + ∆8 transgenic plants
showing high gene expression. Suspensions in either shoot
regeneration medium (SM= MS-based + 1 mg/l BAP and 0.1 mg/l IAA)
or root formation medium (RM= MS-based + 0.1 mg/l IBA) were treated
with different concentrations of acetyl salicylic, ascorbic, caffeic, citric,
jasmonic, nicotinic and salicylic acids to determine the effect of these
elicitors on growth and VLCPUFAs biosynthesis.
1
0.5
0
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0.6
0.4
0.8
0.6
0.4
0.2
0.2
0
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0
0
1
2
3
Conc. (mg/l)
4
Effect of salicylic acid in presence of SM and RM
1
0.8
0.6
0.4
0.2
0
0
2
RM light
4
6
Conc. (m g/l)
RM dark
SM light
8
10
10
SM dark
20
30
40
50
60
Conc. (m g/l)
5
1.2
Weight (g)
3. GC was performed on methylated extracts of freeze dried leaves.
Figure 3: GC profiles of fatty
acid methyl esters in chicory
cv. Pan di Zucchero. Fatty
acids were extracted from a
non-transformed plant (A), and
transgenic plants expressing
∆9 elongase activity (B), both
∆9 elongase and ∆8 desaturase
activities (C) and ∆9 elongase,
∆8 and ∆5 desaturase activities
(D).
1: Linoleic acid.
2: α-Linolenic acid.
3: Eicosadienoic acid.
4: Eicosatrienoic acid.
5: Dihomo-γ-linolenic acid.
6: Eicosatetraenoic acid.
7: Arachidonic acid.
8: Ecosapentaenoic acid.
W e ig h t (g )
1
2. PCR and RT-PCR were performed on DNA and RNA respectively,
extracted from leaves of putatively transformed plants.
1
1.2
1.5
1. Cotyledons from 14d-old seedlings of five cvs. of Cichorium intybus
were transformed by Agrobacterium tumefaciens carrying genes for ∆9
elongase, ∆8 desaturase, ∆5 desaturase and ∆9 elongase + ∆8
desaturase.
1 2
Effect of ascorbic acid in presence of SM amd RM
Effect of acetylsalicylic acid in presence of SM and RM
Methods:
Figure
4:
Effect
of
elicitors on growth of cell
suspensions
generated
from cv. Brussels Witloof
plants
expressing
∆9
together with ∆8 genes.
Conclusions:
• Transgenic plants of chicory were morphologically similar to non-transformed plants.
• ω3 and ω6 fatty acids were produced in transgenic chicory plants.
• Different elicitors affect the growth rate differently in cell suspensions of transgenic plants.
178bp
1 2 3 4 5 6 7 8 9
1 2
3
4
5
500bp
159bp
6
7
8
1C
Acknowledgments:
500bp
175bp
Figure 2: RT-PCR for expression of the ∆9 elongase gene in leaves.
The Egyptian Government for providing a
Higher Degree Scholarship.
Lane 1: postive control, lane 2: negative control. Lanes 3, 5, 6, 8 and 9 are
representative examples of transgenic plants expressing the ∆9 elongase
gene. Lanes 4 and 7 did not show any expression
The Plant Sciences Division, School of
Biosciences, The University of Nottingham
for travel fund.