Hooks M A, Fleming Y, Larson T R, Graham I A
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, UK.
Planta. 1999 Jan;207(3):385-92. doi: 10.1007/s004250050496.
Leaves from transgenic Brassica napus L. plants engineered to produce lauric acid show increased levels of enzyme activities of the pathways associated with fatty acid catabolism (V.A. Eccleston and J.B. Ohlrogge, 1998, Plant Cell 10: 613-621). In order to determine if the increases in enzyme activity are mirrored by increases in the expression of genes encoding enzymes of beta-oxidation, which is the major pathway of fatty acid catabolism in plants, the medium-chain acyl-acyl carrier protein (ACP) thioesterase MCTE from California bay (Umbellularia california) was over-expressed under the control of the cauliflower mosaic virus 35S promoter in Arabidopsis thaliana (L.) Heynh. Arabidopsis was the most suitable choice for these studies since gene expression could be analyzed in a large number of independent MCTE-expressing lines using already well-characterized beta-oxidation genes. Levels of MCTE transcripts in leaves varied widely over the population of plants analyzed. Furthermore, active MCTE was produced as determined by enzymatic analysis of leaf extracts of MCTE-expressing plants. These plants incorporated laurate into triacylglycerol of seeds, but not into lipids of leaves as shown by gaschromatographic analysis of total fatty acid extracts. The expression levels of the beta-oxidation and other genes that are highly expressed during developmental stages involving rapid fatty acid degradation were measured. No significant difference in gene expression was observed among MCTE-expressing plants and transgenic and non-transgenic controls. To eliminate the possibility that post-translational mechanisms are responsible for the observed increases in enzyme activity acyl-CoA oxidase activity was also measured in leaves of MCTE-expressing plants using medium and long chain acyl-CoA substrates. No significant increases in either medium- or long-chain acyl-CoA oxidase activities were detected. We conclude that endogenous beta-oxidation is sufficient to account for the complete degradation of laurate produced in rosette leaves of Arabidopsis expressing MCTE.
经基因工程改造以生产月桂酸的转基因甘蓝型油菜植株的叶片，其与脂肪酸分解代谢相关途径的酶活性水平有所提高（V.A. 埃克莱斯顿和J.B. 奥尔罗格，1998年，《植物细胞》10: 613 - 621）。为了确定酶活性的增加是否与编码β-氧化酶的基因表达增加相对应，β-氧化是植物脂肪酸分解代谢的主要途径，来自加州月桂树（加州桂）的中链酰基-酰基载体蛋白（ACP）硫酯酶MCTE在花椰菜花叶病毒35S启动子的控制下在拟南芥中过表达。拟南芥是这些研究的最合适选择，因为可以使用已充分表征的β-氧化基因，在大量独立的MCTE表达系中分析基因表达。在所分析的植物群体中，叶片中MCTE转录本的水平差异很大。此外，通过对MCTE表达植物的叶片提取物进行酶分析确定产生了活性MCTE。这些植物将月桂酸掺入种子的三酰甘油中，但如总脂肪酸提取物的气相色谱分析所示，未掺入叶片的脂质中。测量了在涉及快速脂肪酸降解的发育阶段高表达的β-氧化和其他基因的表达水平。在MCTE表达植物与转基因和非转基因对照之间未观察到基因表达的显著差异。为了排除翻译后机制导致观察到的酶活性增加的可能性，还使用中链和长链酰基辅酶A底物在MCTE表达植物的叶片中测量了酰基辅酶A氧化酶活性。未检测到中链或长链酰基辅酶A氧化酶活性的显著增加。我们得出结论，内源性β-氧化足以解释在表达MCTE的拟南芥莲座叶中产生的月桂酸完全降解的原因。
