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林业科学 ›› 2017, Vol. 53 ›› Issue (1): 62-69.doi: 10.11707/j.1001-7488.20170108

• 论文与研究报告 • 上一篇    下一篇

超表达牛奶子EutPDS提高番茄果实番茄红素含量

程珍霞, 胡海涛, 杨莉, 王长春, 郭卫东, 杨玲   

  1. 浙江师范大学化学与生命科学学院 金华 321004
  • 收稿日期:2016-01-18 修回日期:2016-07-07 出版日期:2017-01-25 发布日期:2017-03-03
  • 通讯作者: 杨玲
  • 基金资助:
    国家自然科学基金面上项目(31071775);浙江省科技计划项目(2008C24G2030004)。

Overexpression of EutPDS Gene from Elaeagnus umbellata Increases Lycopene Content in Tomato Fruit

Cheng Zhenxia, Hu Haitao, Yang Li, Wang Changchun, Guo Weidong, Yang Ling   

  1. College of Chemistry and Life Sciences, Zhejiang Normal University Jinhua 321004
  • Received:2016-01-18 Revised:2016-07-07 Online:2017-01-25 Published:2017-03-03

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EutPDS基因(GenBank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SlPSY和SlZDS受影响显著上调,同时1个下游基因SlLCY-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。

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

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