Food Lipids: Chemistry, Nutrition, and Biotechnology

esterase in embryos of seed from Umbellularia californica that could conceivably
account for the production of lauric acid. Moreover, that specific enzyme activity
appeared to be missing in similar extracts from canola seed. Encouraged by that
finding, a team of biochemists purified the U. californica lauroyl–ACP thioesterase
protein sufficiently to obtain, by means of an automated protein sequencer, a partial
amino acid sequence. This amino acid sequence allowed the design of synthetic DNA
primers, which were then used with templates made by molecular biologists from
messenger RNA isolated from developing seed of U. californica to generate genespecific
DNA probes. The DNA probes were subsequently used to identify lauroyl–
ACP thioesterase complementary DNA (cDNA) clones from a cDNA bank that was
constructed using, again, messenger RNA (mRNA) from developing seed of U.
californica.
Independently in the same laboratory, another group of molecular biologists
isolated a canola gene specifically expressed at high levels in developing embryos
during the normal period of canola seed development when storage lipids are formed.
This natural B. napus gene was dissected down to the elements necessary to encode
proper gene expression timing and tissue-specific localization within the canola plant.
These ‘‘promoter’’ or genetic expression elements were then combined with the central
portion of the U. californica lauroyl–ACP thioesterase cDNA close corresponding
to the open reading frame encoded by the original naturally occurring messenger
RNA. This synthetic gene was then combined with a second gene (the selectable
marker gene) in a specific manner relative to other DNA signal sequences in a unique
microbe known as a disarmed Agrobacterium tumefaciens.
Plant cell biologists then took the genetically engineered strain of A. tumefaciens
and cultivated it for a brief time with sectioned hypocotyl tissues from germinated
B. napus plantlets. This now-routine method of gene transfer into canola
had to be developed while the biochemists were studying the lauroyl–ACP thioesterase
enzyme in U. californica seed extracts and while molecular biologists were
identifying the gene expression control elements from embryo-specifically expressed
genes in seed of B. napus. After the treatment of B. napus hypocotyl sections with
A. tumefaciens, the bacteria were completely removed and the plant tissues cultured
on a series of different growth media in different containers, to regenerate complete
B. napus canola plants. During this process of regeneration, most of the plantlets
were deliberately killed by addition of an antibiotic. The selectable marker gene that
was linked to the synthetic lauroyl–ACP thioesterase gene in the A. tumefaciens
strain was a synthetic gene designed to detoxify the antibiotic used in these experiments.
Thus the only plants that survived the antibiotic treatment were transgenic
plantlets that had received the kanamycin resistance gene (and almost always the
lauroyl–ACP thioesterase gene as well). This is how the cell biologist can selectively
produce 30 to 300 different transgenic plants without having to sort through
thousands of plants that only might be transgenic. Each transgenic plant coming out
of this process is potentially different and is generally regarded as a different event.
As it turns out, each event tends to be unique in one or several features, most notably,
in this case, with respect to how much lauric acid accumulates in the seed oil.
Progeny from each different event can be grown up and examined for the lauric
acid content in the seed oil, and for other traits as well. As mentioned above, the
transgenes show inheritance like regular canola genes, so gene segregation is possible,
especially when a transgenic canola is crossed with another line.
Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.