| Sweet potato (Ipomoea batatas L. Lam.) has the potential to be engineered to produce or increase specific phytonutrients necessary in undernourished populations of the world. The purpose of this study was to compare the results of a high performance liquid chromatography (HPLC) analysis of carotenoid levels with the gene expression analyses for PSY, LCYb and CHYb, which are the genes encoding the key enzymes phytoene synthase, lycopene beta-ring cyclase, and beta-ring 3 hydroxylase involved in the carotenoid biosynthetic pathway, in orange-fleshed (Beauregard, Hernandez, Georgia jet, Jewel and Carolina Ruby) and yellow-fleshed (Kyukei 97) sweet potato cultivars. The genes were identified using gene-specific primers designed from EST sequences available in GenBank. Total RNA was extracted from each cultivar and the desired sequences were amplified by RT-PCR (reverse transcriptase polymerase chain reaction). Product verification was assessed using a T/A cloning sequencing method. The relative level of gene expression was determined by semi-quantitative RT-PCR. Cloning of the 5'UTR and 3'UTR regions was accomplished using RLM-RACERTM. The main carotenoid detected by HPLC in sweet potato orange-fleshed storage roots was beta-carotene. No zeaxanthin, alpha-carotene, or lutein was detected. In the yellow-fleshed cultivar, only trace amounts of beta-carotene were detected. All three genes were expressed at similar levels in both the orange and yellow-fleshed cultivars, even though the carotenoid level differed dramatically. The LCYb complete coding sequence was obtained, which showed 98% similarity at the amino acid level with a LCYb sequence from the yellow petals of Ipomoea sp. (morning glory). The expression of these key genes does not appear to be correlated with the carotenoid content found in the sweet potato storage roots, suggesting that the regulation of carotenoid accumulation is not occurring at the transcriptional level. |
