Cloning And Analysis Of Eugenol Synthase Gene RCEGS1in Rosa Chinensis ’Pallida’

Eugenol is an important fragrant component in Rosa chinensis’Pallida’, with strong odor of clove. It was reported that coniferyl alcohol served as the substrate of Eugenol synthase (EGS). To clone and analyze eugenol synthase gene in R. chinensis’Pallida’, can not only lay a foundation for the research of synthesis pathway of key synthase genes in rose, and provide candidate genes for rose-scent breeding.A new eugenol synthase gene named RcEGS1was cloned from the petal of R. chinensis’Pallida’by RT-PCR and RACE-PCR, the expression profile of RcEGS1of Rose flower was analyzed by quantitative Real Time PCR in different tissues and different developmental stages. A plant expression vector pBIA31-RcEGSl was constructed by inserting the Open Reading Frame (ORF) of RcEGS1by digestion and ligation and transferred into petunia hybrida plants. These main results were as follows:1.Using PCR degenerate primers designed based on GenBank published the conserved protein sequences of eugenol synthase genes to amplify cDNA fragments by RT-PCR and RACE-PCR, a new eugenol synthase gene named RcEGS1was cloned from the petals of R. chinensis’Pallida’, and its GenBank accession number is JQ522949. The full cDNA was1171bp in length with an open reading frame (ORF) of951bp encoding a protein of317amino acids, has conserved sequences of KQVDVVIS and KIIAAIK. Molecular weight and isoelectric point of the protein was35.64kD and7.36, respectively. Amino acids homology analysis indicated that the RcEGS1and RhEGSl proteins are47.7%identical to each other, and had70%and59%homologies with CbEGS2in Clarkia breweri and PhEGS1in Petunia hybirida, respectively.2. The expression profile of RcEGS1was analyzed by quantitative Real Time PCR in different tissues and different developmental stages of Rose flower. The results showed that RcEGSl expressed specifically in the flower petals and the sepals, and it had a highest transcript level in the petal. In addition, RcEGS1was higher transcript level in the developmental blooming stage, and lower levels in that of bud and senescence stage, and confirmed the same rhythmic with the scent emission.3.The RcEGS1cDNA fragment was recloned into the expression vector pBIA31in Smal and Sail sites. The resulting plasmid pBIA31-RcEGS1was transformed into Trans5α cells. The correct colonies were confirmed by PCR and restriction enzyme digestion of SmaI and SalI. The results showed expression vector pBIA31-RcEGS1was successfully constructed.4. The constructed plasmid pBIA31-RcEGS1was transformed into Trans5α cells. The leaf discs from healthy petunia hybrida were used as explants.7transgenic lines were obtained by PCR.