淡花紫錐菊和文心蘭花色基因功能分析

Abstract

Three major classes of plant pigments are flavonoids/anthocyanins, carotenoids and betalains. In this thesis, the effects of the genes encoding flavonoids and carotenoids on metabolites, and the phenotypic changes in transformants by Agrobacterium-mediated transformation were studied. Agrobacterium-mediated transformation and genetic assay of chalcone synthase in the medicinal plant Echinacea pallida. Echinacea pallida is widely used as an herbal medicine, and is a subject of research in Europe and North America. Micropropagation of Echinacea pallida has been established, but the efficiency of the rooting was low. However, the efficiency of lateral root initiation was increased and the reduction of callus hyperhydricity was improved by supplying gibberellins (GA) instead of auxin. Agrobacterium-mediated transformation was established in Echinacea pallida by modification of the method previously used for E. purpurea (Wang and To, 2004). The most important factor for the transformation of E. pallida was the introduction of a pre-selection phase in which E. pallida leaves were sensitive to antibiotics. The chalcone synthase (CHS) is a key enzyme in the biosynthesis of diverse flavonoids. Here, we investigated the functions of two clusters CHSs, EpaCHS-A and EpaCHS-B, which includes EpaCHS-B1and EpaCHS-B2, isolated from E. pallida to analyze their roles and gene expression in the Petunia CHS transgenic plants. EpaCHS-A and EpaCHS-B1 were found abundantly in petals, whereas EpaCHS-B2 expression was mainly detected in leaves. Nevertheless, the expression of EpaCHSs remained constant in leaves and roots of transgenic E. pallida overexpressing Petunia CHS, while EpaCHS-B2 expression changed in flowers of transgenic plants. In addition, overexpression of Petunia CHS also affected the secondary metabolites in leaves and roots of E. pallida. Modification of flower color by suppressing β-ring carotene hydroxylase genes in Oncidium. Oncidium Gower Ramsey (Onc. Gower Ramsey) is a popular cut flower, but its color is limited to bright yellow. The β-ring carotene hydroxylase (BCH2) gene is involved in carotenoid biogenesis for pigment formation. However, the role of BCH2 in Onc. GR is poorly understood. Here, we investigated the functions of three BCH2 genes, BCH-A2, BCH-B2, and BCH-C2, isolated from Onc. GR to analyze their roles in flower color. RT-PCR expression profiling suggested that BCH2 genes were mainly expressed in flowers. The expression of BCH-B2 remained constant while that of BCH-A2 gradually decreased during flower development. Using Agrobacterium tumefaciens to introduce BCH2 RNA interference (RNAi), we created transgenic Oncidium plants with downregulated BCH expression. In the transgenic plants, flower color changed from the bright yellow of the wild-type to light- and white-yellow. BCH-A2 and BCH-B2 expression levels were significantly reduced in the transgenic flower lips, which make up the major portion of Oncidium flower. Sectional magnification of the flower lip showed that the amount of pigmentation in the papillate cells of the adaxial epidermis was proportional to the intensity of yellow coloration. HPLC analysis of the carotenoid composition of the transgenic flowers suggested major reduction in neoxanthin and violaxanthin pigments. Conclusively, BCH2 expression regulated the accumulation of yellow pigments in Oncidium flowers, and the downregulation of BCH-A2 and BCH-B2 changed the flower color from bright yellow to light- and white-yellow. Functional studies of pigmentation genes in E. pallida and Oncidium by transformation and metabolite analysis show that overexpression of Petunia CHS gene in E. pallida or the repression BCH2 genes in transgenic Oncidium profoundly affects the metabolites of pigment biosynthesis in both transformants.