Abstract
Conjugated linolenic acids are present as major seed oils in several plant species. Punicic acid (or trichosanic acid) is a conjugated linolenic acid isomer containing cis-delta9, trans-delta11, cis-delta13 double bonds in the C(18) carbon chain. Here we report cDNAs, TkFac and PgFac, isolated from Trichosanthes kirilowii and Punica granatum, that encode a class of conjugases associated with the formation of trans-delta11, cis-delta13 double bonds. Expression of TkFac and PgFac in Arabidopsis seeds under transcriptional control of the seed-specific napin promoter resulted in accumulation of punicic acid up to approximately 10% (w/w) of the total seed oils. In contrast, no punicic acid was found in lipids from leaves even when the conjugases were driven under control of the cauliflower mosaic virus 35S promoter. In yeast cells grown without exogenous fatty acids in the culture medium, TkFac and PgFac expression resulted in punicic acid accumulation accompanied by 16:2delta(9cis, 12cis) and 18:2delta(9cis, 12cis) production. Thus, TkFac and PgFac are defined as bifunctional enzymes having both conjugase and delta12-oleate desaturase activity. Furthermore, we demonstrate that 16:2delta(9cis, 12cis) and 18:3delta(9cis, 12cis, 15cis) as well as 18:2delta(9cis, 12cis) are potential substrates for the conjugases to form trans-delta11 and cis-delta13 double bonds.
Highlights
A large number of fatty acid species have been found in plant seed oils
Isolation of cDNAs Encoding FAD2-related Enzymes—Total fatty acids extracted from maturing seeds of T. kirilowii and P. granatum were analyzed by gas chromatography (GC)
We prepared RNA from these materials and isolated four cDNAs from T. kirilowii (TkFad2 and TkFac) and P. granatum (PgFad2 and PgFac) that encode polypeptides related to FAD2
Summary
FAD2-related cDNA Isolation—Total RNA was isolated from maturing seeds of T. kirilowii and P. granatum by the methods of Carpenter et al [13]. Full-length cDNAs of four FAD2-related cDNAs were isolated by PCR amplification with Pyrobest DNA polymerase (Takara Shuzo) using a set of primers corresponding to the sequences in 5Ј- and 3Ј-untranslated regions. Expression of FAD2-related Enzymes in Saccharomyces cerevisiae— The BamHI-SacI fragment of TkFac cDNA released from pGEM-3Z/ TkFac as described above was cloned into the yeast expression vector pYES2 with the galactose-inducible GAL1 promoter (Invitrogen) to generate pYES2/TkFac. pGEM-T Easy/PgFac was digested with EcoRV/SacI and cloned into the plasmid vector pBluescript II (Stratagene), and the PgFac cDNA was released with HindIII/SacI digestion and cloned into pYES2 to generate pYES2/PgFac. The coding regions of TkFad and PgFad were amplified by PCR using Pyrobest DNA polymerase and cloned into pGEM-T Easy as described above.
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