Regulation of Carotenoid Content in Tomato by Silencing of Lycopene β/ε-Cyclase Genes
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 13.8 μg/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 117.3 μg/g and 4.9 to 23.5 μg/g leaf dry weight, respectively. In Lyc-ε RNAi lines, β-carotene content increased, ranging from 195.8 to 290.2 μg/g, while lutein content decreased, ranging from 3.7 to 11.3 μg/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
This research was supported by National Natural Science Foundation of China (30460081) and Scientific Research Fund of Xinjiang BingTuan, China (ZD2007JC06).
- Davuluri GR, Van Tuinen A, Fraser PD, Manfredonia A, Newman R, Burgess D, Brummell DA, King SR, Palys J, Uhlig J, Bramley PM, Pennings HM, Bowler C (2005) Fruit-specific RNAi-mediate suppression of DETl enhances carotenoid andflavonoid content in tomatoes. Nat Biotechnol 23:825–826CrossRefGoogle Scholar
- McGinnis K, Murphy N, Carlson AR, Akula A, Akula C, Basinger H, Carlson M, Hermanson P, Kovacevic N, McGill MA, Seshadri V, Yoyokie J, Cone K, Kaeppler HF, Kaeppler SM, Springer NM (2007) Assessing the efficiency of RNA interference for maize functional genomics. Plant Physiol 143:1441–1451CrossRefPubMedGoogle Scholar