Isolation And Characterization Of Eugenol Synthase Genes In Prunus Mume ’Sanlunyudie’

Floral scent, which is honoured as the soul of flowers, plays a significant role in flavouring industry, pollinators attraction and commercial improvement. Prunus mume is an important traditional flowering tree with typical floral scent in China. As its characteristic compound, eugenol is catalyzed by eugenol synthase with coniferyl acetate as a substrate. To date, studies on floral scent of Prunus mume is still preliminary, and have not gone deep into the molecular metabolism yet. In this thesis,3 eugenol synthase genes were first cloned from Prunus mume’Sanlunyudie’. Then analysis of bioinformatics, expression patterns and transformations were done. Main results are described as follows:1. Three EGS genes designated as PmEGSl, PmEGS2 and PmEGS3, located on the No.2, No.3 and No.6 chromosome,respectively, were first isolated by RT-PCR from fully opened flowers of ’Sanlunyudie’. They contained a complete open reading frame of 927 bp/951 bp/930 bp, encoding 308/316/309 amino acids.The gene sequence numbers are Pm005841, Pm012360, Pm021776. cDNAs and the deduced amino acid sequences all contained the characteristic conserved domains KQVDVVIS and KIIAAIK, and clustered with other EGS genes with high identity based on DNAMAN and homology based on phylogenetic tree derived from Fragaria ananassa, Chinese rose (Rosa hybrida and Rosa chinensis), Ocimum basilicum, Clarkia breweri, Petunia hybrid and etc.2. Flower development was classified into four stages, i.e. flowers with loose calyxes, flowers starting to open, and flowers wilting faded. Meanwhile, flower tissue at the stage of flowers fully open was divided into 3 parts:petals, stamens, sepal+pistil.Quantitative real-time PCR results showed that PmEGS2 and PmEGS3 expressed mainly in petals and stamens. They expressed increasingly all the while until achieved the maximum at stage 4, consistant with the endogenous content and emission regulations of eugenol, which suggested they were probably key genes involved in eugenol synthesis. Meanwhile, the rangeability and multiples of PmEGS2 were much higher than PmEGS3, which represented a stronger expression even a higher combined efficiency of eugenol. Whereas, PmEGSl exhibiting a converse trend may be suppressed by other metabolism pathway.3. Full-length cDNA of PmEGS2 was connected with the expression vector pCAMBIA1304. The recombinant plasmid named pCAMBIA1304-PmEGS2 was transferred into Petunia hybrida’W115’ through Agrobacterium tumefaciens (EHA 105) mediated transformation with leaf disc method. Volatiles of petunia flowers were detected by GC/MS. The result showed that the wild petunias did not contain eugenol while the transgenic petunias contained slightly, indicating that PmEGS2 was involved in the biosynthesis of eugenol.In this thesis, three eugenol synthase genes were isolated from the characteristic scented Prunus mume ’Sanlunyudie’,and preliminary analysis were done on their expression patterns. After the full-length cDNA of PmEGS2 which was significant expressed was transformed to the floral scent model plant Petunia hybrida, the gene was comprehensively studied aiming at initially clarifying the functions and molecular mechanisms of PmEGSs, and providing a theoretical basis for scent-targeted breeding in Prunus species.