中文摘要：

【目的】牛奶子果实中富含番茄红素,八氢番茄红素脱氢酶（PDS）是番茄红素生物合成上游的重要酶。特异性超表达牛奶子Eut PDS基因（Gen Bank登录号：GQ254067）,以期提高番茄果实中的番茄红素含量。【方法】通过RT-PCR方法从牛奶子果实中分离EutPDS基因cDNA及基因组全长,Southern杂交分析基因的拷贝数,专用软件分析此基因及蛋白结构。采用番茄2A11启动子构建果实特异性超表达载体,转化根瘤农杆菌GV3101后,借助农杆菌介导的叶盘法转化至‘中蔬四号’番茄。半定量RT-PCR检测转基因植株果实EutPDS基因的表达,高效液相色谱仪、实时定量PCR分别用于分析转基因番茄果实主要类胡萝卜素组分含量和内源类胡萝卜素代谢相关基因表达的变化。【结果】从牛奶子中克隆的EutPDS基因长度为1 920 bp,含有1 749 bp ORF。其基因组序列无内含子,单拷贝。EutPDS编码1个582个氨基酸的蛋白,该蛋白与其他植物PDS有很高的同源性,具有典型的二核苷酸结合域、类胡萝卜素结合域。采用农杆菌介导方法转化番茄叶盘,PCR检测,潮霉素筛选,成功获得了2个EutPDS转基因番茄株系,其果实颜色较对照更红。半定量分析显示外源EutPDS基因在转基因番茄果实中超表达,实时定量PCR分析表明番茄红素生物合成上游2个内源基因Sl PSY和Sl ZDS受影响显著上调,同时1个下游基因Sl LCY-e的表达则显著下调,使得转基因番茄果实中番茄红素含量为野生型的2倍,但β-胡萝卜素含量无显著变化。【结论】在番茄果实中特异性超表达牛奶子EutPDS基因可有效促进番茄果实中番茄红素的合成和积累。

英文摘要：

【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 Eut PDS（Gen Bank number：GQ254067）driven by fruit-specific promoter.【Method】The full-length c DNA and genomic DNA of Eut PDS gene were isolated from E.umbellata fruit using RT-PCR.The copy number was determined by southern blot hybridization.The structural features of Eut PDS gene and protein were analyzed by using special software.A recombinant plasmid carrying Eut PDS 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 Eut PDS 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 Eut PDS cDNA has 1 920 bp in length and contained a 1 749 bp ORF.The Eut PDS gene was not interrupted by any intron and exists as a single copy in E.umbellata genome.Eut PDS 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 Eut PDS was transformed into tomato leaves by Agrobacterium-mediated method.Two transgenic lines of Eut PDS wereobtained after PCR detection and hygromycin screen.The transgenic fruits were redder than wild type.Semi-quantitative PCR analysis showed that Eut PDS gene was overexpressed in tr