关键词: 牛奶子, 番茄, 番茄红素, 八氢番茄红素脱氢酶基因（PDS）, 载体构建, 遗传转化

Abstract: [Objective] Lycopene has a strong anti-oxidative activity, which could remove free radicals of oxygen to delay the aging of cells of human body. Phytoene desaturase (PDS) is an important upstream enzyme of lycopene biosynthesis. The fruit of Elaeagnus umbellata was found to accumulate high level of lycopene. This study was aimed to enhance lycopene component of tomato(Solanum lycopersicum)by overexpressing EutPDS (GenBank number: GQ254067) driven by fruit-specific promoter. [Method] The full-length cDNA and genomic DNA of EutPDS gene were isolated from E. umbellata fruit using RT-PCR. The copy number was determined by southern blot hybridization. The structural features of EutPDS gene and protein were analyzed by using special software. A recombinant plasmid carrying EutPDS gene driven by fruit-specific promoter 2A11 was constructed. The expression vector was transferred into the Agrobacterium tumefaciens strain GV3101, and then introduced into a tomato variety ‘Zhongshu 4’ by Agrobacterium-mediated leaf disc method. Semi-quantitative RT-PCR was used to analyze EutPDS expression. High performance liquid chromatography and real-time RT-PCR were used to identity the main components of carotenoids and endogenous carotenoid gene expression in tomato fruits of both transgenic lines (OE) and wild-type, respectively. [Result] The cloned EutPDS cDNA has 1 920 bp in length and contained a 1 749 bp ORF. The EutPDS gene was not interrupted by any intron and exists as a single copy in E. umbellata genome. EutPDS encodes a protein of 582 amino acids, which showed a high identity with other plant PDSs. Both a potential dinucleotide-binding motif and the carotenoid-binding domain were identified. The overexpressing vector of EutPDS was transformed into tomato leaves by Agrobacterium-mediated method. Two transgenic lines of EutPDS were obtained after PCR detection and hygromycin screen. The transgenic fruits were redder than wild type. Semi-quantitative PCR analysis showed that EutPDS gene was overexpressed in transgenic tomato fruit. Real-time quantitative PCR analysis demonstrated that two endogenous upstream genes of lycopene biosynthesis, SlPSY and SlZDS, were significantly upregulated while a downstream gene SlLCY-e was significantly downregulated in the fruit of OE-1 compared with the wild-type. Lycopene content in transgenic tomato fruits was nearly twice as much as that in the wild-type, but β-carotene content was not significantly different.[Conclusion] Fruit-specific overexpression of EutPDS gene in tomato can significantly increase the biosynthesis and accumulation of lycopene.

Key words: Elaeagnus umbellata, Solanum lycopersicum, lycopene, phytoene desaturase gene (PDS), vector construction, genetic transformation