Investigations into β-ketoacyl-ACP Synthase III and enoyl-ACP reductase of plant fatty acid synthase

Abstract

An antisense transgenic approach was taken in order to assess the importance of P-ketoacyl-ACP- Synthase III (KAS III) in regulation and initiation of de novo fatty acid synthesis in planta. The transgenic plants were generated in order to show whether alternative pathways exist to initiate fatty acid synthesis if the most direct route is lost. In order to develop such a study, a KAS III cDNA clone of 1622bp was isolated from a Brassica napus embryo library. This clone was used to generate an antisense construct containing a strong double 35S promoter and a CaMV poly A tail. The antisense vector was transformed into both Brassica napus and Arabidopsis thaliana. Sixty-three antisense KAS III lines were generated in Brassica napus, along with 10 transgenic KAS III Arabidopsis thaliana lines. The open reading frame of the KAS III cDNA clone was overexpressed in E.coli to yield a novel protein of 45kDa. It was subsequently purified and used to raise KAS III polyclonal and sera. This provided an immunological tool that could detect KAS III in leaf and seed extracts in wild type Brassica napus. To analyse whether KAS III caused any pleiotropic effects on other FAS components such as down regulation of other FAS enzymes, a quantitative ELISA assay was developed to Enoyl- ACP-Reductase (ENR). This was the first such assay for the detection of ENR in plants and its development has required careful consideration of sample preparation. Levels of KAS III, ENR and (3-Keto-ACP-Reductase (BKR) were assessed in developing leaf of wild type cultivars, along with total fatty acid levels in the growing leaf. Analysis of transgenic KAS III lines was performed in conjunction with the analysis of transgenic sense and antisense ENR Brassica napus that had become available during the study. Transgenic KAS III lines were seen to be slower growing, but were still able to grow to maturity, flower and set seed. This suggests that the KAS FU down regulation may lead to the use of alternative pathways in the synthesis of the initial condensation product acetoacetyl-ACP. Down regulation of ENR in some antisense KAS III lines suggest that a pleiotropic effect may have been exerted as a result of the lowering of KAS III levels. The analysis of the Tl generations for both KAS III and ENR during this study showed that certain lines possessed a phenotype which is characterised by a slower growing plant, where a change of seed morphology was observed along with a change in testa colour and a decrease in overall fatty acid levels in the seed.