Regulation of Carotenoid Content in Tomato by Silencing of Lycopene β/ε-Cyclase Genes

Abstract

To conduct RNAi interference of Lyc-β and Lyc-ε genes, two plant expression vectors were constructed by inserting the intron fragments of the gusA gene into the two target gene fragments, which were designed in anti-sense directions. After the Agrobacterium tumefaciens-mediated transformation, 13 transgenic tomato plants (seven and six for Lyc-β and Lyc-ε, respectively) were obtained, which was further validated by PCR. Real-time PCR revealed that the messenger RNA abundance
of Lyc-β gene and Lyc-ε gene in transgenic tomato plants was significantly reduced to 8.95% and 13.16%, respectively, of the level of the wild-type
plant. Subsequent high-performance liquid chromatography analysis found that transgenic tomato plant had significantly increased
lycopene content, with the highest value of 13.8μg/g leaf dry weight, which was about 4.2-fold that of wild-type plant. Moreover,
Lyc-β and Lyc-ε interference gene effects were observed on downstream products as well. β-Carotene and lutein contents decreased in Lyc-β RNAi lines, ranging from 40.7 to 117.3μg/g and 4.9 to 23.5μg/g leaf dry weight, respectively. In Lyc-ε RNAi lines, β-carotene content increased, ranging from 195.8 to 290.2μg/g, while lutein content decreased, ranging from
3.7 to 11.3μg/g. For total carotenoids, Lyc-β RNAi lines resulted in 2.9-fold decrease, while Lyc-ε RNAi lines yielded 1.7-fold increase in contents when compared to wild-type control. This study demonstrated that RNAi gene
technology is an effective method for enhancing lycopene content in plants.

Keywordsβ-Carotene–Lutein–Lycopene–Lycopene β-cyclase–Lycopene ε-cyclase–RNAi