| Sweet osmanthus also known as sweet scented osmanthus, is one of the ten famous flowers in China, in the garden and spices production occupies an important position. According to its color difference can be divided into four varieties, but the difference among populations is small, mainly yellow and orange red, the lack of blue and other novelty colors, which to a certain extent, limit its ornamental value.Anthocyanin is the most important water soluble pigment in plants, and its most important function is to endow the plant with colorful color, especially the reproductive organs of plants. At present, its synthesis pathways and regulatory mechanisms are fully understood. DFR and CHS gene as the pathway of two key genes have been cloned in a variety of plants, but the formation of sweet scented osmanthus play a regulatory role has not been reported, it is worth further exploration.The full-length cDNA of CHS gene of Sweet olive was cloned which are related to color formation, The characteristics of the gene sequence and evolution relationships were analyzed to provide a theoretical basis for exploring gene function in the form of colour. In this study, a full-length cDNA sequence of CHS gene was obtained from Sweet olive using RT-PCR and RACE. The gene nucleotide and amino acid sequences were analyzed by using NCBI and DNAMAN and a phylogenetic tree was constructed by using DNAMAN software directly, which will help us analyze the gene evolutionary relationships among different species. Quantitative PCR results showed that, OfCHS gene in in petals and leaves of Osmanthus fragrans were expressed, and in the leaves of relative expression of flowering period petals expression 38 times,with flowering, expression quantity presents the obvious first increased and then decreased trend, and in Osmanthus fragrans varieties, at the beginning of flowering stage expression was the highest, and then decreased gradually.Full-length gene was cloned from Sweet olive successfully, which is displayed as Sweet olive CHS gene, which is 1383 bp in full length and include 5′URT of 48 bp and 3′ URT of 162 bp, has an open reading frame of 1173 bp encoding a deduced polypeptide of 391 amino acids. Sequence analysis revealed that this gene has three active site, Cysl64、His303和Asn336 and two decisions substrate specificity of phenylalanine residues Phe215、Phe265. OfCHS has two characteristics of polypeptide sequence of CHS synthase superfamily: RFMMYQQGCFAGGTVLR and GVLFGFGPGL. Blast sequence showed that similarity between Sweet olive and Olive oil was up to 83 percent and was at around 72%-76% with the other dicots. OfCHS gene belongs to CHS family, phylogenetic analysis showed that the gene cluster has a distinct species characteristics, Sweet olive has a near relationship to Labiatae, which was up to 76%, and it was in line with plant taxonomy,and laid the foundation to explore the mechanism of Of CHS in the formation of the color lineA full-length cDNA of a Dihydroflavonol-4-Reductase(DFR) gene was cloned from sweet osmanthus named OfDFR(KR604812). Sequence analysis showed that the length of OfDFR gene was 1214 bp,and has an open reading frame of 1131 bp which encoded 376 amino acids. It was predicted that the molecular mass of this protein was 99.68 kD,and pI was 4.98. Analysis of functional domain showed that the OfDFR protein included two conserved amino acid sites, belonged to the NADB Rossmann superfamily. Sequence blasted and phylogenetic analysis indicated the DFR had a higher sequence conservation among different species,while sweet osmanthus OfDFR exhibited the highest sequence similarity to Forsythia intermedia Fi DFR. Quantitative real-time PCR analysis demonstrated that the OfDFR gene was expressed both in leaves and flowers and the transcript was significantly higher in leaves than that in petals. During the different floral stages the expression of OfDFR gene increased at first and then decreased. Besides,These results established the basic foundation for the further study on the functions of OfDFR color formation.Two genes were successfully constructed with PBI121 expression vector, named as PBIOfCHS, PBI-OfDFR, and the GV3101 was transformed into Agrobacterium by freezing and thawing method. Arabidopsis thaliana was infected by inflorescence infection method. |
