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

To conduct RNAi interference of Lyc-beta and Lyc-epsilon 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-beta and Lyc-epsilon, respectively) were obtained, which was further validated by PCR. Real-time PCR revealed that the messenger RNA abundance of Lyc-beta gene and Lyc-epsilon 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 13.8 mu g/g leaf dry weight, which was about 4.2-fold that of wild-type plant. Moreover, Lyc-beta and Lyc-epsilon interference gene effects were observed on downstream products as well. beta-Carotene and lutein contents decreased in Lyc-beta RNAi lines, ranging from 40.7 to 117.3 mu g/g and 4.9 to 23.5 mu g/g leaf dry weight, respectively. In Lyc-epsilon RNAi lines, beta-carotene content increased, ranging from 195.8 to 290.2 mu g/g, while lutein content decreased, ranging from 3.7 to 11.3 mu g/g. For total carotenoids, Lyc-beta RNAi lines resulted in 2.9-fold decrease, while Lyc-epsilon 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.