Proceedings
of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini
Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1
Poster
Abstract - 2.11
Expression
of a plant D9 lipid
desaturase gene in the Tobacco plastidial genome
Lenzi
P., Scotti N., Craig W., Monti L., Grillo S., Cardi T.
CNR-IMOF, Research Institute
for Vegetable and Ornamental Plant Breeding, via Università 133, 80055
Portici
plastid transformation,
lipids, metabolic engineering, biopharmaceuticals
The transformation of the plastidial
genome of higher plants offers many attractive features. The precise insertion
of (trans)genes occurs through homologous recombination, thereby eliminating
gene silencing and/or cosuppression effects. This permits an exceptionally high
level of transgene expression (up to approx 50% of TSP) due to naturally high
gene copy numbers. Subsequent downstream processing is assisted as plastids are
also the natural site of production and/or accumulation of several compounds of
commercial interest. Single-step metabolic engineering is now a reality as
plastids are capable of processing polycistronic mRNAs, allowing the expression
of operons containing heterologous genes, whilst the environment is protected
as natural transgene containment occurs due to null or minimal plastid
transmission in pollen of most crop plants.
Current research in our laboratory
includes the optimisation of plastid transformation protocols in various
Solanaceous crops (see companion abstract of Craig et al.), using the biolistic
approach with leaf explants and the PEG/electroporation approach with
protoplasts. Our present interests encompass genes for the manufacture in plant
plastids of compounds useful for human health and agriculture, such as
polyunsaturated fatty acids (PUFAs), isoprenoids produced through the plastidic
pathway, and vaccines for human viruses (HIV-1, HPV).
The D9 lipid desaturase catalyzes the
conversion of stearic acid into oleic acid, a key step for the further
production of unsaturated fatty acids both in the cytosol and in the plastid.
In higher plants, the D9 gene is nuclear, but the enzyme is translocated to the
latter organelle. The gene encoding a plant D9 desaturase, cloned downstream of
a selectable marker gene in dicistronic constructs, has successfully been
relocated to the plastome of tobacco plants via biolistic transformation.
Correct insertion in the plastid genome has been confirmed by PCR and Southern
analyses. Homoplasmic plants were obtained after one or two regeneration
cycles. Northern analysis showed the presence of two primary transcripts
corresponding to the dicistronic and monocistronic mRNAs. The assessment of
protein accumulation and the effect on plant development is currently under
way.