cDNA cloning and expression of Brassica napus enoyl-acyl carrier protein reductase in Escherichia coli
Rent the article at a discountRent now
* Final gross prices may vary according to local VAT.Get Access
The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition. During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly. We describe a rapid and simple purification procedure for the plastidlocalized NADH-dependent enoyl-acyl carrier protein reductase from developing B. napus seed, based on its affinity towards the acyl carrier protein (ACP). The purified protein was N-terminally sequenced and used to raise a potent antibody preparation. Immuno-screening of a seed-specific λgt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones. DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane. Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B. napus NADH-dependent enoyl-ACP reductase. Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B. napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B. campestris. Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.
- Bloch, K, Vance, D: Control mechanisms in the synthesis of saturated fatty acids. Annu Rev Biochem 46: 263–298 (1977).
- Bradford, MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).
- Caughey, I, Kekwick, RGO: The characteristics of some components of the fatty acid synthetase system in the plastids from the mesocarp of avocado (Persea americana) fruit. Eur J Biochem 123: 553–561 (1982).
- Cottingham, IR, Austin, A, Sidebottom, C, Slabas, AR: Purified enoyl-[acyl-carrier protein] reductase from rape seed (Brassica napus) contains two closely related polypeptides which differ by a six-amino-acid N-terminal extension. Biochim Biophys Acta 954: 201–207 (1988).
- Dellaporta, SL, Woods, J, Hicks, JB: A plant DNA minipreparation version II. Plant Mol Biol Rep 1(4): 19–21 (1983).
- Gubler, U, Hoffman, BJ: A simple and very efficient method for generating cDNA libraries. Gene 30: 195–200 (1983).
- Harwood, JL, Walsh, MC, Walker, KA: Enzymes of fatty acid synthesis. Meth Plant Biochem 3: 193–216 (1990).
- Henikoff, S: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: 351–359 (1984).
- Laemmli, UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
- McKeon, T, Stumpf, PK: Stearoyl-acyl carrier protein desaturase from safflower seeds. Meth Enzymol 71: 275–281 (1981).
- McKeon, T, Stumpf, PK: Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. J Biol Chem 257: 12141–12147 (1982).
- Nakamura, Y, Yamada, M: Long chain fatty acid synthesis in developing castor bean seeds I. The operation of the path from acetate to long chain fatty acids in a subcellular particulate system. Plant Cell Physiol 15: 37–48 (1974).
- Olsson, G: Species crosses within the genus Brassica II. Artificial Brassica napus L. Hereditas 46: 351–386 (1960).
- Rock, CO, Cronan, Jr, JE: Solubilization, purification, and salt activation of acyl-acyl carrier protein synthetase from Escherichia coli. J Biol Chem 254: 7116–7122 (1979).
- Rock, CO, Garwin, JL: Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein. J Biol Chem 254: 7123–7128 (1979).
- Rock, CO, Cronan, Jr, JE: Improved purification of acyl carrier protein. Anal Biochem 102: 362–364 (1980).
- Saiki, RK, Gelfand, DH, Stoffel, S, Scharf, SJ, Higuchi, R, Horn, GT, Mullis, KB, Erlich, HA: Primer directed enzymatic amplification of NDA with a thermostable DNA polymerase. Science 239: 487–491 (1988).
- Sambrook, J, Fritsch, EF, Maniatis, T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
- Sanger, F, Nicklen, S, Coulson, R: DNA sequencing with chain terminating inhibitors. Proc Natl Acad USA 74: 5463–5467 (1977).
- Shimakata, T, Stumpf, PK: Purification and characterization of β-ketoacyl-[acyl-carrier-protein] reductase, β-hydroxyacyl-[acyl-carrier-protein] dehydrase, and enoyl-[acyl-carrier-protein] reductase from Spinacia oleracea leaves. Arch Biochem Biophys 218: 77–91 (1982).
- Silva, de, J, Loader, NM, Jarman, C, Windust, JHC, Hughes, SG, Safford, R: The isolation and sequence analysis of two seed-expressed acyl carrier protein genes from Brassica napus. Plant Mol Biol 14: 537–548 (1990).
- Slabas, AR, Sidebottom, CM, Hellyer, A, Kessell, RMJ, Tombs, MP: Induction, purification and characterization of NADH-specific enoyl-acyl carrier protein reductase from developing seeds of oil seed rape (Brassica napus). Biochim Biophys Acta 877: 271–280 (1984).
- Slabas, AR, Harding, J, Hellyer, A, Sidebottom, C, Gwynne, H, Kessell, R, Tombs, MP: Enzymology of plant fatty acid biosynthesis. Dev Plant Biol 9: 3–10 (1984).
- Slabas, AR, Cottingham, IR, Austin, A, Hellyer, A, Safford, R, Smith, CG: Immunological detection of NADH-specific enoyl-ACP reductase from rape seed (Brassica napus) — induction, relationship of α and β polypeptides, mRNA translation and interaction with ACP. Biochim Biophys Acta 1039: 181–188 (1990).
- Slabas, AR, Cottingham, I, Austin, A, Fawcett, T, Sidebottom, CM: Amino acid sequence analysis of rape seed (Brassica napus) NADH-enoyl ACP reductase. Plant Mol Biol 17: 911–914 (1991).
- Studier, FW, Rosenberg, AH, Dunn, JJ, Dubendorff, JW: Use of T7 RNA polymerase to direct expression of cloned genes. Meth Enzymol 185: 60–89 (1990).
- U, N: Genome analysis in Brassica with special reference to the experimental formation of Brassica napus and peculair mode of fertilization. Jap J Bot 7: 389–452 (1935).
- Volpe, JJ, Vagelos, PR: Saturated fatty acid biosynthesis and its regulation. Annu Rev Biochem 42: 21–60 (1973).
- Wakil SJ, Mizugaka M, Shapiro M, Weeks G In: Bolis L, Pethica BA (eds) Membrane Models and the Formation of Biological Membranes, pp. 122–137. North-Holland Publishing Co., Amsterdam.
- Weaire, PJ, Kekwick, RGO: The synthesis of fatty acids in avocado mesocarp and Cauliflower bud tissue. Biochem J 146: 425–437 (1975).
- Weaire, PJ, Kekwick, RGO: The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids. Biochem J 146: 439–445 (1975).
- Ykema, A, Verbree, EC, Nijkamp, HJJ, Smit, H: Isolation and characterization of fatty acid auxotrophs from the oleaginous yeast Apiotrichum curvatum. Appl Microbiol Biotechnol 32: 76–84 (1989).
- cDNA cloning and expression of Brassica napus enoyl-acyl carrier protein reductase in Escherichia coli
Plant Molecular Biology
Volume 17, Issue 4 , pp 895-909
- Cover Date
- Print ISSN
- Online ISSN
- Kluwer Academic Publishers
- Additional Links
- acyl carrier protein
- Brassica napus
- enoyl-ACP reductase
- fatty acid synthesis
- seed development
- nuclear-encoded chloroplast proteins
- Industry Sectors