Molecular Cloning Of CPS Gene In Chrysanthemum Cinerariaefolium And Its Genetic Transformation In Tobacco

Chrysanthemum cinerariaefolium, a perennial herbaceous plant of the Compositae, is the raw material plant for producing natural pyrethrins which serves as high effective, wide used, low toxic and pollution-free pesticide. Along with the enhancement of people's awareness in health and environmental protection, there is an increasing market demand in pyrethrins, especially in those developed countries. Because of the low content of pyrethrins in natural Chrysanthemum cinerariaefolium, it's very important to research the biosynthesis and regulation mechanism of pyrethrins in order to improve its yield to meet the need of the increasing demand.Chrysanthemyl diphosphate synthase (CPS), which catalyze the formation of trimethylene construct, is an important enzyme involved in the biosynthesis of natural pyrethrins. In this paper, a C. cinerariaefolium cultivar named YUNCHU NO.1 was used as experiment material. Total RNA was extracted from the buds of YUNCHU NO.1 and the CPS gene was cloned by RT-PCT. Then plant expression vectors carrying CPS gene were constructed and introduced into Agrobacterium tumefaciens strain GV3101. Subsequently sense and anti-sense CPS genes were transferred into tobacco by Agrobacterium-mediated method. Study on the transgenic plants expressing sense and anti-sense CPS genes might provide some beneficial information for the genes's usage in production of pyrethrins in genetic modified plants. The results were as follows:①The RNA extracted from Chrysanthemum cinerariaefolium buds was used for isolating CPS gene by RT-PCR. The cDNA of CPS contained a 1 185 bp ORF encoding a 395 amino acids constituted protein. Bioinformatic analysis showed that the predicted CPS had a putative chloroplast location signal peptide,and two Asp-rich motifs,which exist in the members of prenyltransferase family typically.②The PCR product of CPS gene was purified and randomly inserted into pDH51 by blunt ligation. And the intermediate vectors pDH-CPSs and pDH-CPSas were successfully constructed by enzyme digestion and electrophoresis.③The CPS Chimeric genes 35SP-CPS-35ST and 35SP-CPSas-35ST were digested from those two intermediate vectors by restriction enzyme and were inserted into cloning site of plant expression vector pCAMBIA1301. By enzyme digestion and electrophoresis analysis, the plant expression recombinant pCAMBIA-CPS and pCAMBIA-CPSas contained reporter gene GUS were successfully constructed.④The recombinant plasmids pCAMBIA-CPSs and pCAMBIA-CPSas were introduced into Agrobacterium tumefaciens strain GV3101 by freezing-thawing method. Through screening on the medium containing kanamycin and the PCR identification of positive clones, it was confirmed that the recombinant plasmids were successfully introduced into GV3101 respectively.⑤The engineering bacteria GC3101 containing pCAMBIA-CPSs and pCAMBIA-CPSas plasmids was incubated with leaves of tobacco plants. Selected tith 50 mg/L Kan and cultured on differentiation medium, regeneration tobacco plants were obtained. The results of genomic DNA PCR and GUS histochemical staining showed that the sense and anti-sense CPS genes were successfully introduced into tobacco. The total RNA was extracted from the pCAMBIA-CPSs transgenic plants and the results of RT-PCR showed that CPS gene expressed normally in tobacco leaves.⑥To detect the chlorophyll and carotenoid contents in transgenic tobacco leaves, spectrophotometer method was used. The results showed that the content of carotenoids significantly decreased in tobacco overexpressing of heterologous Pyrethrum CPS gene, while there is little influences on carotenoids content in tobacco by antisense CPS gene; CPS gene had no effect on chlorophyll content in transgenic tobacco.