Isolation and characterization of two Brassica napus embryo acyl-ACP thioesterase cDNA clones.
Acyl-ACP thioesterases are involved in regulating chain termination of fatty acid biosynthesis in plant systems. Previously, acyl-ACP thioesterase purified from Brassica napus seed tissue has been shown to have a high preference for hydrolysing oleoyl-ACP. Here, oligonucleotides derived from B. napus oleoyl-ACP thioesterase protein sequence data have been used to isolate two acyl-ACP thioesterase clones from a B. napus embryo cDNA library. The two clones, pNL2 and pNL3, contain 1642 bp and 1523 bp respectively and differ in the length of their 3' non-coding regions. Both cDNAs contain open reading frames of 366 amino acids which encode for 42 kDa polypeptides. Mature rape thioesterase has an apparent molecular weight of 38 kDa on SDS-PAGE and these cDNAs therefore encode for precursor forms of the enzyme. This latter finding is consistent with the expected plastidial location of fatty acid synthase enzymes. Northern blot analysis shows thioesterase mRNA size to be ca. 1.6 kb and for the thioesterase genes to be highly expressed in seed tissue coincident with the most active phase of storage lipid synthesis. There is some sequence heterogeneity between the two cDNA clones, but overall they are highly homologous sharing 95.7% identity at the DNA level and 98.4% identity at the amino acid level. Some sequence heterogeneity was also observed between the deduced and directly determined thioesterase protein sequences. Consistent with the observed sequence heterogeneity was Southern blot data showing B. napus thioesterase to be encoded by a small multi-gene family.
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Department of Biological Sciences, University of Durham, UK.
Woolner
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Hellyer
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Slabas
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Safford
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001937:Brassica / Q000196:embryology / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D017343:Genes, Plant; D008969:Molecular Sequence Data; D005810:Multigene Family; D015345:Oligonucleotide Probes / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D010940:Plant Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013869:Thiolester Hydrolases / Q000235:genetics*
D017931:DNA Primers; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D010940:Plant Proteins; D012333:RNA, Messenger; D013869:Thiolester Hydrolases; C018748:oleoyl-(acyl-carrier-protein) hydrolase