JXB Advance Access originally published online on January 30, 2004
Journal of Experimental Botany, Vol. 55, No. 397, pp. 787-789, March 1, 2004
© 2004 Oxford University Press
GENE NOTE |
Isolation and expression pattern of two putative acyl-ACP desaturase cDNAs from Bassia scoparia
Received 15 September 2003; Accepted 25 November 2003
1 Long Ashton Research Station, Long Ashton, Bristol BS41 9AF, UK
2 Rothamsted Research, Harpenden, Herts AL5 2JQ, UK
3 Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK
* To whom correspondence should be addressed at Rothamsted. Fax: +44 (0)1582 763010. E-mail: johnathan.napier{at}bbsrc.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionSupplementary dataReferences |
|---|
The seed lipids of some higher plants contain unusual fatty acids with potentially valuable non-food uses. Seeds of Bassia scoparia contain one such monounsaturated fatty acid, 16:1
5. This fatty acid can be used for the production of an insect oviposition pheromone, which is potentially valuable in the control of the mosquito Culex quinquefasciatus, a vector of West Nile virus. Previous work has established that a number of unusual monounsaturated fatty acids are produced by variant forms of the ubiquitous acyl-ACP desaturases. The isolation and initial characterization of two putative acyl-ACP desaturases from B. scoparia, one of which is seed-specific, suggests that such a variant enzyme occurs in this species. Key words: Acyl-ACP desaturase, Bassia scoparia, Culex quinquefasciatus, oviposition pheromone, unusual fatty acid, West Nile virus.
|
Introduction |
|---|
|
TopAbstractIntroductionSupplementary dataReferences |
|---|
Higher plants, unlike animals, display a wide range of chemical diversity in the fatty acids they collectively synthesize, with currently over 300 different fatty acids identified in the Plant Kingdom, compared with a much smaller number in animals. These ‘unusual’ plant fatty acids are distinct from those which occur in the majority of plant seed tissue, namely palmitic, oleic, linoleic, and linolenic acids. Many of these unusual fatty acids have valuable properties, either as neutraceuticals or for industrial purposes. Monounsaturated fatty acids that have the double bond occurring in a position other that that of the
9 position can be classed as unusual fatty acids. Bassia scoparia (syn. Kochia scoparia, commonly known as Burning Bush or Summer-cypress) is a plant that produces one such unusual and potentially useful unusual monounsaturated fatty acid. Along with a three other 5-desaturated fatty acids (18:15; 18:25,9; 18:35,9,12) this plant also accumulates the monounsaturated fatty acid 16:15 in a seed-specific manner, at around 5% of total seed fatty acids (Kleiman et al., 1972).
This particular C16 monounsaturated fatty acid has the specific use of being a part of a potential control system for the mosquito Culex quinquefasciatus (Olagbemiro et al., 1999). This insect is the major vector species of the filarial worm Wuchereria bancrofti, and is also responsible for transmitting arboviruses such as West Nile virus (Lanciotti et al., 1999). This mosquito has very specific requirements for its larval habitat, and in the correct conditions maturing eggs secrete an oviposition pheromone that is a major olfactory cue to gravid female mosquitoes of the same species to lay their eggs in the same body of water. Combined with a juvenile-hormone-type insecticide, such a semiochemical could potentially be used as an effective control method of this mosquito as a disease vector (Olagbemiro et al., 1999). Whilst conventional chemical synthesis of the oviposition pheromone is possible, it can also be synthesized from the 16:15 in Bassia scoparia in a simple two-step process, to yield (5R, 6S)- 6-acetoxy-5-hexadecanolide (the active pheromone). This latter process has the advantage of being considerably cheaper, as well as utilizing a renewable resource in the form of the 16:15 substrate.
The first desaturation step in plant fatty acid biosynthesis is carried out by soluble acyl-ACP desaturases, located in the plastid. The usual substrate for acyl-ACP desaturases is stearoyl-ACP, resulting in the production of 18:19-ACP (oleoyl-ACP). Previous work from Shanklin and colleagues established that variant acyl-ACP desaturases were responsible for the production of the majority of unusual monounsaturated fatty acids observed in higher plants. Examples include the 4-palmitoyl-ACP desaturase from Coriandrum sativum, the 6-palmitoyl-ACP desaturase from Thunbergia alata and the 9-myristoyl-ACP desaturase from Pelargonium hortorum (Cahoon et al., 1992, 1994; Shultz et al., 1996). Importantly, it was observed that expression of these unusual variant ACP-desaturases was normally restricted primarily to seeds; this is in agreement with many other studies which have reported the accumulation of unusual plant fatty acids only in the triacylglycerols of seed lipids. As part of these research efforts to provide a sustainable and environmentally-benign source of chemical feedstocks to facilitate the synthesis of the Culex oviposition pheromone (Olagbemiro et al., 1999), genes encoding the (presumptive) variant Bassia scoparia 5-palmitoyl-ACP desaturase were sought, with the long-term goal of using such a gene to direct heterologously the synthesis of the monoenoic fatty acid.
Bassia scoparia (Chenopodiaceae) was grown from seed, and the genomic DNA isolated from the leaves of the mature plant using the Qiagen DNeasy kit (Qiagen). Three highly degenerate oligonuleotide primers designed to the conserved motifs of previously characterized acyl-ACP desaturases were synthesized for DNA amplification; two forward primers, D1 (GARGARGCNCTNCCNCANTA), D2 (GCNTGGCANGCNGA RGARAAY), and one reverse, R1 (RCANACRTANTCYTGNGC). The initial PCR was carried out using the D1 and R1 primers, and the resulting band of approximately 750 bp re-amplified using the D2 and R1 primers. Two closely migrating products of similar size, approximately 620–650 bp, were amplified and these were cloned and sequenced. Two different sequences, both with high identity with soluble acyl-ACP desaturases, were thus identified. These genomic sequences were used to design primers for cDNA-RACE for both of the putative acyl-ACP desaturases.
RNA was extracted from developing B. scoparia seeds using the Qiagen RNeasy kit (Qiagen), and this was used as the template for amplification, using the Marathon RACE kit (Clonetech). The 3' and 5' ends of each gene was successfully cloned and sequenced, and the information used to design primers to the terminal 5' (ATG) and 3' (stop) of each deduced desaturase open reading frame. Using these primers the full-length cDNA of each gene was cloned and sequenced; these sequences are available from GenBank (AF315599
[GenBank]
and AF315600
[GenBank]
). One of the putative acyl-ACP desaturases, designated K1, (AF315600
[GenBank]
) had a greater level of identity with previously characterized 9-stearoyl-ACP desaturases, while the other putative acyl-ACP desaturase (AF315599
[GenBank]
), designated KN, had a higher level of identity with previously characterized variant acyl-ACP desaturases. There was found to be 64.2% identity between the two B. scoparia desaturases at the amino acid level. The DNA and deduced amino acid %-similarities with other ACP-desaturases is shown in Table 1.
|
Total RNA was extracted from B. scoparia leaf, stem, root, and developing seed tissue. This was resolved on a 1.2% agarose gel containing 7% formaldehyde and transferred onto Hybond-N+ nylon membrane. KN was probed with 300 bp of the 3' end of the KN clone, while K1 was probed with the 5' RACE product of the K1 gene; both probes contained gene-specific untranslated regions. The northern analysis of both these genes (Fig. 1) shows that while the K1 appears to be highly expressed in the seed tissue it is also present in the leaf and root tissue, while KN is only expressed at low levels in the seed, and not anywhere else. The whole K1 gene was also used to probe the KN blot to check for cross-hybridization; however, no band was detected (not shown). Thus, it appears that the K1 transcript is expressed in both photosynthetic and non-photosynthetic tissues as well as developing seeds, whereas KN is only detected in seeds. These expression patterns imply that the K1 transcript is likely to encode the ubiquitous
9-stearoyl-ACP desaturase, whereas the KN transcript is a potential candidate for the 5-palmitoyl-ACP desaturase. The KN transcript is present (at relatively low levels) only in developing seeds, mirroring the seed-specific accumulation of 16:15 fatty acids. Moreover, the deduced amino acid sequence of KN contains specific heterogeneity at positions previously shown to be determinants of region- and substrate- (i.e. chain-length) specificity; in particular, residues A204 and A211 of the KN sequence which are co-linear with residues T181 and G188 in the castor 9-stearoyl-ACP desaturase. Mutagenesis of these amino acids in the castor 9-stearoyl-ACP desaturase resulted in an altered substrate-specificity towards palmitoyl-ACP (Cahoon et al., 1997b; Whittle and Shanklin, 2001). (A full sequence comparison can be found at Journal of Experimental Botany online.) It is also of interest to note that phylogenetic analysis of ACP desaturase sequences groups the B. scoparia KN sequence with all previous known examples of variant soluble ACP desaturases, whereas K1 segregates with 9-stearoyl-ACP desaturase sequences (HM Whitney, O Sayanova, JA Pickett, JA Napier, unpublished observations, see also Sperling et al., 2003). Initial attempts to express the KN open reading frame in E. coli did not result in any desaturase activity (Whitney, 2002). However, the authors believe that KN is very likely to encode the variant acyl-ACP desaturase responsible for 16:15 synthesis.
|
In conclusion, two sequences have been identified from Bassia scoparia with high homology to soluble acyl-ACP desaturases. Sequence and expression analysis indicates that whilst one cDNA (K1) is likely to encode a
9-stearoyl-ACP desaturase, the second is an excellent candidate for the 5-palmitoyl-ACP desaturase present in seeds of this plant. |
Supplementary data |
|---|
|
TopAbstractIntroductionSupplementary dataReferences |
|---|
Figure S1 is a comparison of the deduced amino acid sequences of acyl-ACP desaturases and can be found at Journal of Experimental Botany online.
|
Acknowledgements |
|---|
Long Ashton Research Station (1903–2003) and Rothamsted Research receive grant-aided support from BBSRC (UK). HMW was the recipient of a BBSRC studentship. We are extremely grateful for the help and advice of John Shanklin (Brookhaven, NY).
|
References |
|---|
|
TopAbstractIntroductionSupplementary dataReferences |
|---|
Cahoon EB, Shanklin J, Ohlrogge JB. 1992. Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco. Proceedings of the National Academy of Sciences, USA 89, 11184–11188.
Cahoon EB, Cranmer AM, Shanklin J, Ohlrogge JB. 1994. 6 hexadecenoic acid is synthesized by the activity of a soluble 6 palmitoyl-acyl protein desaturase in Thunbergia alata endosperm. Journal of Biological Chemistry 269, 27519–27526.
Cahoon EB, Coughlan SJ, Shanklin J. 1997a. Characterization of a structurally and functionally diverged acyl-acyl carrier protein desaturase from milkweed seed. Plant Molecular Biology 33, 1105–1110.[CrossRef][Web of Science][Medline]
Cahoon EB, Lindqvist Y, Schneider G, Shanklin J. 1997b. Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position. Proceedings of the National Academy of Sciences, USA 94, 4872–4877.
Cahoon EB, Shah S, Shanklin J, Browse J. 1998. A determinant of substrate specificity predicted from the acyl-acyl carrier protein desaturase of developing cat’s claw seed. Plant Physiology 117, 593–598.
Kleiman R, Rawls MH, Earler FR. 1972. cis-5-monoenoic fatty acids in some Chenopodiaceae seed oils. Lipids 7, 494–495.[CrossRef]
Lanciotti RS, Roehrig JT, Deubel V, et al. 1999. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 286, 2333–2337.
Olagbemiro TO, Birkett MA, Mordue (Luntz) AJ, Pickett JA. 1999. Production of (5R,6S)-6-acetoxy-5-hexadecanolide, the mosquito oviposition pheromone, from the seed oil of the summer cypress plant, Kochia scoparia (Chenopodiaceae). Journal of Agricultural Food Chemistry 47, 3411–3415.[CrossRef]
Shultz DJ, Cahoon EB, Shanklin J, Craig R, Cox-Foster DL, Mumma RO, Medford JI. 1996. Expression of a 9-14:0-acyl carrier protein fatty acid desaturase gene is necessary for the production of 
5 anacardic acids found in pest-resistant geranium (Pelargoniumxhortorum). Proceedings of the National Academy of Sciences, USA 93, 8771–8775.
Sperling P, Ternes P, Zank TK, Heinz E. 2003. The evolution of desaturases. Prostaglandins Leukot Essential Fatty Acids 68, 73–95.[CrossRef][Web of Science][Medline]
Whitney HM. 2002. Isolation and characterization of desaturase genes from Kochia scoparia. PhD thesis, University of Bristol.
Whittle E, Shanklin J. 2001. Engineering 9-16:0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor 9-18:0-ACP desaturase. Journal of Biological Chemistry 276, 21500–21506.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||