| Purple yam (Dioscorea alata L.) is one of Dioscoreaceae plant have been called ’purple ginseng’because of a plenty of anthocyanins in the tuber and the health benefits nourishing the stomach, lung and kidney. Anthocyanins play a very important role on resistance to bio-and abiotic stresses, as also as on the horticulture, makeup and food industries. Anthocyanins biosynthesis pathway have deeply been studied in the plants such as arabidopsis, petunia, grape, and tobacco. Hence the study on purple yam is relatively plain leading to a great limitation ahead of the research on anthocyanins biosynthesis in yam. And four aspects were probed into purple yam in this study, expression profiles of related genes, finding the key gene of anthocyanins synthesis, expression profiles of transcriptional factors, and cloning of the related genes. All of them contributed to the research and further exploration on anthocyanins biosynthesis system in yam.The materials of purple and white cultivars (Dioscorea alata L.) origined from Taizhou, Zhejiang province were planted on field and greenhouse. In order to find out the key point resulting the difference of coloration between white and purple yam, expression profiles of anthocyanins biosynthesis genes PAL, F3H, F3’H, F3’5’H, DFR, ANS and UFGT during the development of yam were analysized. The results showed that ANS and UFGT expression levels were significantly higher in young leaves and tubers of purple yam (P<0.01). The stressed experiment by high temperature and drought in greenhouse reduced anthocyanins content, up-/down-regulated others genes including ANS except for UFGT. Which suggested UFGT might be the key point of anthocyanins biosynthesis in purple yam.Expression analysis of biosynthesis genes and transcriptional factors TT1, EGL3, TTG1 and TTG2 during anthocyanins accumulation in purple yam tubers showed the early biosynthesis genes (EBGs) PAL, F3H, F3’H and F3’5’H had higher expression levels in the early period and hardly in later periods of tuber development. While the expression profiles of late biosynthesis genes (LBGs) DFR, ANS and UFGT showed consistency with anthocyanins accumulation rate both in early and late period, so did as the expression profiles of TT1, EGL3, TTG1 and TTG2. That suggested the LBGs and transcriptional regulators assisted to anthocyanins accumulation throughout the development of tuber, while the EBGs only contributed to the early period. What’s more, F3’H and F3’5’H that expressed higher quantities in white plant tissues were not only contributed to anthocyanins accumulation but also the fundamental development of plants morphogenesis.The full coding nucleotide sequence lengths of DαF3H and DαFLS1 genes were obtained from purple yam with the 3’-RACE,5’-RACE and general PCR methods. The GenBank accession numbers were KF561995 and KJ022640 respectively for DαF3H and DαFLS1. Both of the deduced proteins were belonged to the 20G-Fe (Ⅱ) dioxygenase superfamily. Phylogenetic trees of the deduced amino acids sequences of DαF3H and DαFLS1 showed they were both in single branches, which suggested they both had a far evolutionary distance from other plants. Expressional analysis of DαF3H and DαFLS1 indicated they expressed specialy highest in young tissues. The dark treated experiment designed according to the GT-1 binding site contained in DαF3H promoter sequences showed DαF3H was regulated by light and enhanced the relationship of DαF3H and anthocyanins biosynthesis and accumulation.This study is a important means of pointing out the key gene controlling the coloration of purple yam, demonstrating the anthocyanins accumulation in yam tuber being a dynamic process, showing F3’H and F3’5’H expression levels are in accordance with plant development. Moreover the cloning of DaF3H and DaFLSl genes supplied theoretical foundation and technical guidance for further studies on anthocyanins biosynthesis in purple flesh tuber of yam. |
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