Separation Of Fragrance-related Genes Of Osmanthus Fragrans By Using CDNA-AFLP Technology

Sweet osmanthus (Osmanthus fragrans) is the representatives specie of oleaceae-osmanthus, well-known for its green and fragrance value.In this study, using cDNA-AFLP technique, sweet osmanthus flower buds were taken as the material to separate of differential segments during the process of osmanthus fragrans formation, obtained 6 TDFs which were possible identified to be associated with the formation of sweet osmanthus by cloning, sequecing and BLAST analysis. This laid a foundation for revealing flavor sweet-scented metabolic pathway of osmanthus and provided a new approach to the genetic improvement of flower scents. These results are as follows:1. Establishing the isolation technology system adaptable for total RNA in osmanthus flowersTotal RNA in osmanthus flowers buds was isolated by improved hot borate method, improved Tris-boric acid method and kit, By comparison, the results showed that total RNA by kit, with good quality and high purity and concentrations, was suits for further testes.2. Separation of differential segments during the process of osmanthus fragrans formation by cDNA-AFLPIn this study, a fingerprinting approach--amplified fragment length polymorphism (AFLP) was done using selective base pairs, including 16 EcoR I primers and 16 Mse I primers, 256 primer combinations had been used, which 160 primer combinations were selected. 100 differential expression transcript-derived fragments(TDFs) were obtained, of which 50 TDFs were cloned and sequenced. The results revealed that, using database homology searches, 31 TDFs were putative genes and 17 TDFs were unknown genes. According to the analysis of 31TDFs, 6 TDFs may be associated with fragrans, named as 2OGox2,C4H1,CCD4,AACT,CFAT, LOX.3. The separation of 3′sequence of CFAT, LOX by RT-PCRTwo differential segments, CFAT and LOX, were selected for their possible Correlativity with fragrans, their 3′sequences were gained by RT-PCR, cloned, sequenced and spliced, the length of sequences were 862bp and 1588bp separately.These studies, for metabolism approach and genetic engineering of fragrans, will establish a basis.