Chrysanthemyl Diphosphate Synthase:Trichome Specific Promoter Characterization, Overexpression Regulates Pyrethrins Biosynthesis In Tanacetum Cinerariifolium And Resolving Relationships Within Chrysanthemum

Tanacetum cinerariifolium (Asteraceae) commonly known as pyrethrum is an economically important for the production of a group of insecticidal secondary metabolites collectively called pyrethrins. Pyrethrins produce all over the aerial parts of the plant but are occurred at high concentrated in the flower achenes. Glandular trichomes on the outside of flower achenes and secretory duct-like structures inside the pericarp have been suggested to be involved in the biosynthesis of pyrethrins. The first committed steps in the biosynthesis of pyrethrins are the formation of chrysanthemyl diphosphate from two molecules of dimethylallyl diphosphate (DMAPP) by chrysanthemyl diphosphate synthase (TcCDS). TcCDS evolved relatively recently after a gene duplication from an ancestral farnesyl diphosphate synthase (FDS) gene. The similarity between the CDS and FDS gene families are as high as 70% based on a sequence from Artemisia annua and closely related species within the Asteraceae family. In the present research, we cloned and characterized trichome specific TcCDS promoter in T.cinerariifoilum. This is the first report to provide a basis for further experiments in T. cinerariifolium and characterization of cis-elements in relation to the various transcription factors, environmental and developmental conditions that may affect the expression of TcCDS and biosynthesis of pyrethrins. It can be used for trichome specific metabolic engineering to improve the quality and yield of secondary metabolites in plants. In previous studies, pyrethrins content was improved by conventional breeding or with chemical treatments such as ethrel, chlormequat chloride and paclobutrazol but the improvements have been relatively limited compared to what can potentially be achieved by means of metabolic engineering of the biosynthetic pathway. No one has been taken initiative to increase pyrethrins content through metabolic engineering. On the other hands, in artemisia lot of work proved that overexpressing some of the artemisinin biosynthetic enzyme genes to be an effective way to increase the amount of artemisinin. In this study, we have taken initiative to develop transgenic T. cinerariifolium with higher levels of pyrethrins by over expression strategy with TcCDS gene. Then, we analyzed the genetic variation of the CDS gene within Chrysanthemum and to test the utility of the CDS gene for the construction of phylogenetic trees. This is essential and significant in the management of germplasm collections and efficiently utilizing the biodiversity of this genus for breeding new chrysanthemum cultivars. On the other hands TcCDS gene sequences have helped us to develop a better understanding of the evolutionary history of chrysanthemum and its wild relatives. The main results are summarized below:1. In this study, we have done molecular cloning and characterization of the trichome specific chrysanthemyl diphosphate synthase promoter from Tanacetum cinerariifolium. An 1128 bp TcCDS promoter fragment was cloned from pyrethrum genomic DNA. The sequence contained cis-elements predicted to be responsive to different hormones, light, and environmental stresses. To characterize the promoter it was fused to the reporter genes β-glucuronidase (GUS) and transformed into tobacco (Nicotiana tabacum). GUS staining of tobacco showed that the TcCDS promoter was exclusively expressed in the glandular secretory trichomes (GSTs). The findings will support research on factors influencing the accumulation of pyrethrins and can be used for trichome-specific metabolic engineering of plants to ensure minimal adverse effects on plant growth and development.2. To investigate the contribution of key enzyme chrysanthemyl diphosphate synthase {TcCDS) to the overall control of the pyrethrins biosynthesis pathway in plants. TcCDS gene was overexpressed in T. cinerariifolium plants to study the effects on pyrethrins. The transgenic lines developed from putative transgenic regenerants were evaluated using NPTII gene probe. The transgenic lines showed positive bands of NPTII gene. Real-time quantitative PCR result indicated that transgenic lines had shown higher expression levels of TcCDS compare to control. The HPLC analyses showed that the pyrethrins contents were significantly increased in these transgenics. One of the transgenic lines, T2-4, was found to contain 3.0 fold higher pyrethrins than the non-transgenic plant. The results demonstrate the regulatory role of TcCDS in pyrethrins biosynthesis.3. To study the molecular evolution and phylogenetic utility of the chrysanthemyl diphosphate synthase (TcCDS) gene, resolving relationship within Chrysanthemum. A 1000 bp genomic DNA fragment of the CDS gene was isolated by nested PCR. About 16 wild species collected from gene banks of China, Japan, and the Netherland were sequenced. The result showed that, the values of nucleotide substitutions per valid common sites intron regions were much higher than those of the exon region indicating the presence ot evolutionary constraints. Molecular evolutionary analysis revealed that there are considerable differences in the number of synonymous and non synonymous substitutions per site. Phylogenetic trees were built by bootstrap NJ methods. The result of phylogenetic analysis showed that, CDS gene could provide a good simple information and clearly showed the three interspecific hybrids, C. x coreanum, C.x pekinense andC.x indicum shared the same group and closest to two Japanese species and C. indicum. The Chinese ancestral species, C. indicum is the most closely related to modern hybrids. TcCDS gene sequences have provided better resolution and stronger support of the evolutionary history of chrysanthemum and its wild species.