Mechanism Of Flower Color Variation And CDNA-AFLP Analysis Of2Cultivars With Different Flower Color In Osmanthus Fragrans

Osmanthus fragrans, one of ten famous flowers in China belongs to Osmanthus. It is a beautiful andaromatic plant and has a long history of2500years. The famous flower contains more than50fragransingredients and has been used in food, spice, medicine etc.In this paper, we used3cultivars with different color: Zi Yingui(light yellow), Jingui(yellow), andChenghong Dangui(orange red) to uncover the mechanism of flower color variation, using cDNA-AFLPtechnique to screen different expression genes, which can help the study of regulation of the flower color.The main results as follows:1. We used HPLC technique to analyze the carotenoids content of3cultivars, Zi Yingui, Jingui andChenghong Dangui, in5different flowering stages and confirm the main carotenoids in the3cultivars.The results showed that it can accumulate only a little β-carotene in Zi Yingui petals, lutein andβ-carotene in Jingui petals, a lot of α-carotene and β-carotene in Chenghong Dangui petals. The differentflower color of3cultivars was due to the various carotenoids and their content, and this illuminated thechemical mechanism of flower color variation in Osmanthus fragrans.2. Using homology-based cloning method, we cloned the partial or full length genes of carotenoidsmetabolic pathway and4main genes of anthocyanin metabolic pathway（CHS, CHI, DFR and ANS）inOsmanthus fragrans. The results of sequence alignment and clustering analysis with some other speciesshowed that the cloned partial of full length genes had a close relationship with some other species. Theresults of RT-PCR analysis confirmed that the different content of carotenoids was due to the differentialexpression level of genes in carotenoids metabolic pathways. The differential expression level ofOfCCD4gene between Zi Yingui and Chenghong Dangui was the main reason, resulting in thedifferential carotenoids content and various flower color in the2cultivars. Because the DFR and ANSwere almost no expression in the2cultivars, we think that it can not synthesis anthocyanin in Osmanthusfragrans and the flower color is only due to the carotenoids content.3. The methylation analysis of the CG island in OfCCD45’ upstream and the first codon showed that the total methylation level in the promoter region of Chenghong Dangui is significant higher than that in ZiYingui and it has no methylation site in both region of the first condon. In the flower of ChenghongDangui, high methylation level impressed gene expression and the low expression of OfCCD4gene wasdue to the high methylation level in the promoter region. It has two W-box palindromic structure in thefront of OfCCD4promoter region, so the gene may be regulated by WRKY transcription factors.4. We cloned3WRKY transcription factors from petal of Osmanthus fragrans. The RT-PCR analysisshowed that expression pattern of the3genes were consistent with OfCCD4. The yeast co-transformedinto by2recombinant plasmid, pB42AD-OfWRKY3and pLacZi-W-box, can activate expression ofβ-galactosidase and the result of yeast one hybrid suggested that OfWRKY3regulate expression ofOfCCD4gene.5. Using cDNA-AFLP technique, we analyzed the differential expression profile between the2cultivars,and screened114genes including genes of substance metabolism, energy metabolism, stress resistance,relation to senescence, transcription factor, and unknown genes. Therefore it has a greater differentialexpression profile between the2cultivars, which involved several metabolic pathways and signaltransduction pathways.