Isolation and characterization of two Brassica napus embryo acyl-ACP thioesterase cDNA clones

Abstract

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.

Metabolic engineering of fatty acid biosynthesis in Indian mustard (Brassica juncea) improves nutritional quality of seed oil

“…8) of B. juncea upon introduction of MbFatB gene encoding a FatB type acyl-ACP thioesterase can be explained by assuming that heterologously expressed MbFatB in seed tissue of B. juncea competes with the predominantly expressed endogenous BjFatA (Loader et al 1993;Pathak et al 2004) for availability of the acyl-ACP substrate. The MbFatB, having preferential thioesterase activity (Jha et al 2006) towards C16:0-ACP available earlier in the metabolic pathway of fatty acid biosynthesis (Fig.…”

“…First, the over-expression of a novel acyl-ACP thioesterase of FatB encoded by the MbFatB gene from Diploknema (Madhuca) butyracea (Jha et al 2006) was explored to reduce the VLCUFA content by early termination of the acyl chain-elongation process, expecting to increase C16 and C18 FAs in the seed-oil. It is worth mentioning here that the endogenous FatB thioesterase is poorly expressed in seed tissues of B. juncea (Jha et al 2006) that predominantly expresses FatA thioesterase (Loader et al 1993;Pathak et al 2004). As a second strategy, double-strand or hair-pin (hp) RNA-mediated silencing of the endogenous fatty acid elongase (BjFAE) gene encoding 3-ketoacyl CoA synthase (KCS), required for erucic acid synthesis from C18:1 acyl pool (Bao et al 1998;Domergue et al 1999;Gupta et al 2004), was employed.…”

No Induction of ?-oxidation in leaves of Arabidopsis that over-produce lauric acid

“…During transport from the plastid the fatty acid is reactivated to a usable form by ligation to coenzyme A for further biosynthetic reactions, such as chain elongation and/or incorporation into membrane lipids or triacylglycerol for storage in lipid bodies during the development of oilseeds (reviewed in Browse and Sommerville 1991). Oilseed species, which include Arabidopsis thaliana (Voelker et al 1992;DoÈ rmann et al 1995) and Brassica napus (Hellyer and Slabas 1990;Loader et al 1993) possess acyl-ACP thioesterases (thioesterases) primarily for the cleavage of fatty acids longer than 16 carbons, which comprise the vast majority of fatty acids produced in these species (Gibson et al 1994). When a thioesterase more speci®c for medium-chain fatty acids, such as laurate (C12), was expressed under the control of the seed-speci®c napin promoter in Arabidopsis (Voelker et al 1992) or the cauli¯ower mosaic virus (CaMV) 35S promoter in B. napus (Eccleston et al 1996), signi®cant amounts of laurate were incorporated into triacylglycerol.…”

Techniques of cell type-specific transcriptome analysis and applications in researches of sexual plant reproduction

“…During embryogenesis, a singlecelled zygote follows a defined pattern of cell division and differentiation to form a mature embryo. To investigate the developmental expression of mRNAs in embryos, the isolated embryos from many species (e.g., soybean, rapeseed, barley and rice) were first used for the cDNA library construction and the isolation of embryo specific transcripts (Goldberg et al, 1981;Loader et al, 1993;Ranford et al, 2002;Ito et al, 2004). Hand-dissected embryo proper and suspensor cells from the globular embryos of the scarlet runner bean were used for differential screening to identify suspensor-specific mRNAs (Weterings et al, 2001).…”