牛奶子果实成熟过程中类胡萝卜素组成及代谢酶基因的克隆与表达
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摘要
番茄红素(lycopene)是一种链式类胡萝卜素,脂溶性胡萝卜素的异构体,具有11个共轭双键和2个非共轭双键。由于其共轭双键的独特分子结构,番茄红素在猝灭单线态氧、消除自由基等方面具有较高的活性。大量研究表明,番茄红素具有一定的防治人类某些慢性疾病和心血管疾病的作用。近期的研究发现,即使低浓度的番茄红素也可以有效的抑制结肠癌细胞的生长。由于番茄红素在维持人类健康方面的重要作用,人们对富含番茄红素的食品和营养品的需求急剧增长。因而,番茄红素新种质资源的挖掘、开发,利用遗传育种方法特别是采用转基因手段调控番茄红素的合成,选育番茄红素含量高的番茄品种或其它经济作物,扩大番茄红素的来源具有十分重要的意义。
美国科学家发现,牛奶子(Elaeagnus umbellate Thunb.)浆果中番茄红素含量相当于普通番茄的18倍,最新研究显示高达75 mg/100 g鲜重。参与番茄红素生物合成的酶,多数通过基因工程的手段克隆出了相应的基因,并成功通过遗传工程技术提高了番茄、马铃薯中番茄红素的含量。但是,有关牛奶子果实中大量番茄红素积累机制的研究还是空白。为此,我们首次分离得到7个牛奶子类胡萝卜素合成基因——Ggps、Psy、Pds、Zds、Lcy-b、Lcy-e、Bch和两个内参基因——Gapdh、Actin。为深入研究牛奶子果实中类胡萝卜素合成基因与番茄红素积累的关系,我们运用实时定量PCR和RT-PCR技术对基因的表达情况进行分析。结论如下:
①牛奶子果实中大量番茄红素的积累是类胡萝卜素代谢途径各基因协同作用的结果。上游4个番茄红素合成基因——Ggps、Psy、Pds和Zds的上调,下游番茄红素代谢基因——Lcy-b和Bch的下调,尤其是Lcy-e基因的沉默可能在番茄红素大量积累中起重要作用。
②牛奶子果实成熟过程中,尽管叶黄素合成途径中番茄红素环化酶基因Lcy-e沉默,叶黄素(包括α-胡萝卜素)却存在相当的含量,原因可能是Lcy-e基因在牛奶子果实发育早期表达而产生大量的叶黄素,然后随着果实的增大叶黄素的单位含量逐渐下降。
③Pds基因开放阅读框(ORF)为1749 bp,编码582个氨基酸、分子量约65.2 kD、等电点约8.12的蛋白质,牛奶子Pds基因全长DNA序列不含任何内含子。系统发育分析说明牛奶子与杏属亲缘关系较近。
④牛奶子类胡萝卜素合成基因Pds与Psy均是单拷贝的。
Lycopene, a red linear carotenoid and an isomer of fat-soluble carotene with 11 conjugated double-bonds and two non-conjugated double-bonds, is the most potent antioxidant from plant sources and exhibits the highest physical quenching rate constant with singlet oxygen. Lycopene could reduce the risk of chronic diseases such as cardiovascular diseases and different forms of cancer, and inhibite the growth of human colon cancer cells even at low concentration. Due to the important role of lycopene in human health, consumer demand for lycopene-rich food and nutraceutical products is growing. Therefore, the development for new idioplasm resources of lycopene was very significant. Heredity breeding, especially regulating lycopene biosynthesis and modifying tomato variety or other industrial crops to rich source of lycopene with transgenic method is useful.
By far the most promising is autumn olive fruit, in which the lycopene is about 17 times higher than that of tomato, and reaches up about 75 mg/100 g fresh weight. The pathway of carotenoid biosynthesis in higher plants is illustrated. Carotenoid biosynthesis and its regulation have been studied in various plant species. But little is still known about the molecular mechanism of lycopene biosynthesis in autumn olive fruit. In this case, we cloned seven carotenogenic genes Ggps, Psy, Pds, Zds, Lcy-b, Lcy-e, Bch, and two control genes Gapdh and Actin from autumn olive. All of these carotenogenic genes were evaluated at transcript level in order to determine which of them played a major role in massive lycopene accumulation during fruit ripening. Characterization of the full-length Pds was also studied. The results are as follows:
①The lycopene accumulation in autumn olive fruit was highly regulated by the coordination of the expression among carotenogenic genes. The massive lycopene accumulation in autumn olive fruit was concomitant with the up-regulation of upstream genes of lycopene synthesis (Ggps, Psy, Pds and Zds), and down-regulation of downstream genes (Lcy-b and Bch), and in particular with the silence of Lcy-e throughout fruit ripening.
②The formation of lutein was feasible before stage A. The level of lutein decreased progressively from stage A to stage D probably due to a dilution effect caused by the enlargement of the fruit. This theory can be used to explain the presence of lutein though Lcy-e is silent.
③The full-length cDNA of Pds contains an ORF of 1749 bp encoding a polypeptide of 582 amino acids, and was not interrupted by any intron. In silico analysis of the predicted amino acid sequence showed a pI of 8.12 and molecular mass of 65.2 kD.
④The genomic DNA of autumn olive contains a single copy of Pds and Psy.
美国科学家发现,牛奶子(Elaeagnus umbellate Thunb.)浆果中番茄红素含量相当于普通番茄的18倍,最新研究显示高达75 mg/100 g鲜重。参与番茄红素生物合成的酶,多数通过基因工程的手段克隆出了相应的基因,并成功通过遗传工程技术提高了番茄、马铃薯中番茄红素的含量。但是,有关牛奶子果实中大量番茄红素积累机制的研究还是空白。为此,我们首次分离得到7个牛奶子类胡萝卜素合成基因——Ggps、Psy、Pds、Zds、Lcy-b、Lcy-e、Bch和两个内参基因——Gapdh、Actin。为深入研究牛奶子果实中类胡萝卜素合成基因与番茄红素积累的关系,我们运用实时定量PCR和RT-PCR技术对基因的表达情况进行分析。结论如下:
①牛奶子果实中大量番茄红素的积累是类胡萝卜素代谢途径各基因协同作用的结果。上游4个番茄红素合成基因——Ggps、Psy、Pds和Zds的上调,下游番茄红素代谢基因——Lcy-b和Bch的下调,尤其是Lcy-e基因的沉默可能在番茄红素大量积累中起重要作用。
②牛奶子果实成熟过程中,尽管叶黄素合成途径中番茄红素环化酶基因Lcy-e沉默,叶黄素(包括α-胡萝卜素)却存在相当的含量,原因可能是Lcy-e基因在牛奶子果实发育早期表达而产生大量的叶黄素,然后随着果实的增大叶黄素的单位含量逐渐下降。
③Pds基因开放阅读框(ORF)为1749 bp,编码582个氨基酸、分子量约65.2 kD、等电点约8.12的蛋白质,牛奶子Pds基因全长DNA序列不含任何内含子。系统发育分析说明牛奶子与杏属亲缘关系较近。
④牛奶子类胡萝卜素合成基因Pds与Psy均是单拷贝的。
Lycopene, a red linear carotenoid and an isomer of fat-soluble carotene with 11 conjugated double-bonds and two non-conjugated double-bonds, is the most potent antioxidant from plant sources and exhibits the highest physical quenching rate constant with singlet oxygen. Lycopene could reduce the risk of chronic diseases such as cardiovascular diseases and different forms of cancer, and inhibite the growth of human colon cancer cells even at low concentration. Due to the important role of lycopene in human health, consumer demand for lycopene-rich food and nutraceutical products is growing. Therefore, the development for new idioplasm resources of lycopene was very significant. Heredity breeding, especially regulating lycopene biosynthesis and modifying tomato variety or other industrial crops to rich source of lycopene with transgenic method is useful.
By far the most promising is autumn olive fruit, in which the lycopene is about 17 times higher than that of tomato, and reaches up about 75 mg/100 g fresh weight. The pathway of carotenoid biosynthesis in higher plants is illustrated. Carotenoid biosynthesis and its regulation have been studied in various plant species. But little is still known about the molecular mechanism of lycopene biosynthesis in autumn olive fruit. In this case, we cloned seven carotenogenic genes Ggps, Psy, Pds, Zds, Lcy-b, Lcy-e, Bch, and two control genes Gapdh and Actin from autumn olive. All of these carotenogenic genes were evaluated at transcript level in order to determine which of them played a major role in massive lycopene accumulation during fruit ripening. Characterization of the full-length Pds was also studied. The results are as follows:
①The lycopene accumulation in autumn olive fruit was highly regulated by the coordination of the expression among carotenogenic genes. The massive lycopene accumulation in autumn olive fruit was concomitant with the up-regulation of upstream genes of lycopene synthesis (Ggps, Psy, Pds and Zds), and down-regulation of downstream genes (Lcy-b and Bch), and in particular with the silence of Lcy-e throughout fruit ripening.
②The formation of lutein was feasible before stage A. The level of lutein decreased progressively from stage A to stage D probably due to a dilution effect caused by the enlargement of the fruit. This theory can be used to explain the presence of lutein though Lcy-e is silent.
③The full-length cDNA of Pds contains an ORF of 1749 bp encoding a polypeptide of 582 amino acids, and was not interrupted by any intron. In silico analysis of the predicted amino acid sequence showed a pI of 8.12 and molecular mass of 65.2 kD.
④The genomic DNA of autumn olive contains a single copy of Pds and Psy.
引文
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