Functional Research Of EutPSY, LCY-e And Isolation Of EutUGP From Autumn Olive Fruit

Lycopene, the most antioxidative substance which widely distributes in the nature is a functional nature pigment. It has various physiological functions including anticancer, antioxidation, mutation inhibition, DNA protection and so on. Moreover, lycopene can regulate cell metabolism and cholesterol metabolism. The lycopene consumption for a healthy diet is growing.The fruit of autumn olive (Elaeagnus umbellate Thunb.) is rich in many human health-related nutrients, such as amino acids, minerals, vitamins, soluble sugars, lycopene and other substances. In particular, the lycopene is about18times higher than that of tomato. Therefore, study on the process of lycopene accumulation and the metabolistic mechanism of sugar and organic acids is useful for exploration of new lycopene germplasm resources, breeding of high lycopene tomato varieties or other cash crops. Our former research showed that the transcript level of EutPSY (phytoene synthase) was up-regulated and EutLCY-e (lycopene s-cyclase) was silence throughout autumn olive fruit ripening. EutLCY-e and EutPSY may be concomitant with the massive lycopene accumulation in autumn olive fruit. Proteomic analysis showed that EutUGP (UDP-glucose pyrophosphorylase) and other three related enzymes of glucose metabolism were up-regulated throughout autumn olive fruit ripening. So EutUGP may involve with the soluble sugar accumulation in autumn olive fruit. The results of this study are as follows:1. EutPSY over-expression vector driven by E8fruit-specific promoter and SILCY-e RNAi vector were constructed. Both plasmids were transformed into tomato (Solanum lycopersicum L. cv Zhongshu No.4). 2. Transformation into tomato was conducted by leaf disc regeneration system. EutPSY over-expression and SILCY-e RNAi vectors were introduced into sterile tomato cotyledons by Agrobacterium-mediated transformation. The best preculture medium of tomato cotyledons was Murashige and Skoog medium(MS)+2mg/L trans-Zeatin+vitamine C+folic acid. The medium of tomato cotyledons co-cultured with Agrobacterium was MS+2mg/L trans-Zeatin+vitamine C+folic acid+acetosyringone. The off-Agrobacterium medium of tomato cotyledons was MS+2mg/L trans-Zeatin+vitamine C+folic acid+cefotaxime, and the selective medium of transgenic plants was MS+2mg/L trans-Zeatin+vitamine C+folic acid+cefotaxime+hygromycin B.3. Tomato transgenic lines were acquired and the foreign gene was confirmed by PCR for the expected bands.There were four transgenic lines including one line of EutPSY over-expression and three lines of SILCY-e RNAi.4. We have cloned the full length of EutUGP gene using rapid amplification of cDNA ends. The full-length cDNA of EutUGP contained an ORF of1431bp encoding a polypeptide of476amino acids, and was interrupted by18introns. The full length DNA of EutUGP was6221bp. A953bp promoter of EutUGP was isolated by genome walking kit. And it contained two sucrose responsive elements and many other cis-acting elements.