Epsilon Archive for Student Projects

Abstract

Synthetically produced pheromones have evolved as non-toxic alternatives to conventional pesticides in modern agriculture. Pheromone traps are used for mass trapping and mate-disruption of many pest insects resulting in substantial yield increases and reduced pesticide use. However, due to high production costs and toxic residues from the chemicals required for the production, cheaper
and more environmentally friendly alternatives are being investigated. One strategy is to make plants produce pheromone precursors or pheromone components by genetic transformation. One promising candidate is Camelina sativa, an oil crop with suitable genetic, physiological, and
agronomic traits and a high proportion of seed oil (triacylglycerol), 30-40%, of its seed weight.

In this study, four mutant lines of C. sativa, were analysed. The lines were transformed with genes from Cuphea species and insect desaturases to produce pheromone precursors that can be further transformed into pheromones. The lines produce various amounts of their target fatty acids, and to improve these results and increase the proportion of the target fatty acids in the future, the focus of this study has been to map the flow and bottlenecks of the triacylglycerol (TAG) biosynthesis in each line. Lipid analyses of seed TAG showed accumulation of the target fatty acids varying from 7% to 43% of the TAG content. In three lines the target fatty acid also accumulated in the membrane lipid phosphatidylcholine (PC) which might be harmful for the plant. One of the pheromone lines,
14:1, though, produced 24% of total TAG content consisted of the pheromone precursor fatty acid 14:1 (Z9) without accumulation in the membrane lipids, which is promising. In enzyme assays of microsomal fractions, the enzymatic activity could be followed through the TAG synthesis by
detecting the radiolabelled flow in the lipid intermediates from glycerol-3-phosphate (G3P) into TAG. The results showed that the added 18-carbon CoAs was the most efficient substrate in the mutant lines as well as in the WT control. The added 14 and 16-carbon acyl-CoAs were detected in
very low amounts in TAG and were also accumulated in the membrane lipids for all lines. We can see that the acylation of 18-carbon-CoAs into the intermediate phosphatidic acid (PA) is working, indicating that the plants endogenous enzymes glycerol-3-phosphate acyltransferase (GPAT) and
lysophosphatidylacyl-transferase (LPAT) are active, but for the 14-carbon and 16-carbon fatty acids the amount of radiolabel in PA is very low. Camelina GPAT and LPAT thus seems to have difficulties to transfer these target fatty acids even though there is likely an abundance of the acyl-
CoAs in the endoplasmic reticulum (ER).

To further test/increase the efficiency of the pheromone precursor fatty acids some experiments could be done. Adding enzymes (mainly LPAT that has a strict preference for unsaturated acyl-CoAs, preferably 18 carbon long) from plants with high levels of 14-16 carbons might increase the level of the target fatty acid in the transgenic lines. For future experiments, [14C] lysophosphatidyl acid (LPA), PA or DAG (with different fatty acids attached) could be added instead of G3P, to get more detailed information about bottlenecks in the transgenic lines.