Functional Research Of The Carotenoids Biosynthesis Gene In Autumn Olive

Lycopene is a kind of natural terpenoids,and has strong antioxidative activity,which could help to remove oxygen free radicals and reduce the incidence of cardiovascular disease or some cancers.Humans are incapable to synthesize lycopene on their own,unless obtain through diet.Eighty-five percent of lycopene is gained from tomato and its processed products.Therefore,researches on gene functions involved in carotenoid biosynthesis metabolism will provide theoretical basis for using biological engineering to regulate the synthesis of lycopene.Autumn olive is a perennial woody plant,and lycopene content of its ripe fruits can be up to 54mg/100g.Expressions of related genes involved in carotenoids synthesis in different periods during ripening of autumn olive fruit were analyzed.The results showed EutPSY(phytoene synthase gene)and EutPDS(phytoene desaturase gene)were significantly upregulated as the fruit matures,but EutLCY-e(lycopene epsilon-cyclase gene)was silenced.Therefore,we hypothesized that the high accumulation of lycopene in autumn olive fruit may be closely related these three genes.In this paper,the functions of EutPSY,EutPDS and SILCY-e were studied by using tomato genetic transformation method,meanwhile EutLCY-e genes was also cloned.1.An over-expression vector of EutPDS gene driven by 2A11 fruit-specific promoter was successfully constructed.2.EutPSY,EutPDS over-expression vector,and SILCY-e RNAi vector were transformed into tomato through Agrobacterium-mediated genetic transformation.After hygromycin selection,three lines of EutPSY over-expression,one line of EutPDS over-expression,and four lines of SILCY-e interference were obtained.3.The RT-PCR and qRT-PCR analysis showed that the EutPDS gene expressed in EutPDS line,but not in the wild-type,the expression of EutPSY in the three over-expression lines was higher than in the wild-type,and the expression of SILCY-e gene in SILCY-e interfering lines was lower than in the wild-type.These results indicated that exogenous gene was successfully expressed in transgenic tomato.4.By HPLC determination of lycopene and ?-carotene content in the transgenic and wild-type tomato fruit,we found that ?-carotene content in the three over-expression lines of EutPSY was significantly increased by 1.7-2.3 times.Lycopene content in three EutPSY over-expression lines and one EutPDS over-expression line was increased by 1.8-2.6 times,but there was no significant change in the interference lines.5.The full length of EutLCY-e gene was cloned by using rapid amplification of cDNA ends(RACE)method.The EutLCY-e gene contains a 1725 bp open reading frame encoding 574 amino acids,and shares a high homology in plants.The full length DNA of EutLCY-e is 4145 bp,and contains 10 introns.