【作者】 李殷；

【导师】 唐克轩；

【作者基本信息】 复旦大学 ， 遗传学， 2009， 博士

【摘要】 作为一种重要的脂溶性抗氧化物质,维生素E(生育酚)在人类和动物的日常膳食中起着不可或缺的作用。维生素E只能在光合自养植物中合成,人类的日常所需摄取白植物源性的食物或药物补充。近来,借助于基因组学和分子生物学技术,维生素E生物合成途径的关键酶基因已在模式生物拟南芥(Arabidopsis thaliana)和蓝藻(Synechocystis sp. PCC 6803)中得以确认并克隆,由此廓清了植物维生物E生物合成途径的结构。本文以高等植物拟南芥维生素E生物合成途径关键酶编码基因为基础,通过转基因方法对该途径进行遗传操作,围绕维生素E生物合成途径进行了两个方向、四个具体内容的研究。一个方向是以模式生物拟南芥为平台,着力于评价生物合成途径各位点关键酶基因对维生素E合成产生的影响,为提升作物维生素E总量或改变作物维生素E组成提供有效的策略,并在蔬菜作物生菜中进行初步的验证,成功获得了大幅度提升维生素E含量和组成的高营养价值的作物品系；另一方向由关键酶和代谢途径作为切入点,研究维生素E在植物生理中的作用,观察分析了维生素E与维生素C之间的关系,并且探讨了维生素E对植物逆境防御的影响。四个具体研究内容概述如下：1)拟南芥遗传背景下维生素E生物合成途径关键酶作用评价以模式生物拟南芥为平台,在同一遗传背景下单独转化维生素E生物合成途径五个关键酶基因(hppd、hpt、mpbq mt、tc和γ-tmt),通过分析过量表达株系维生素E的含量和组成,对各个关键酶在维生素E生物合成途径中的作用加以评价。实验发现,转化维生素E生物合成途径的关键酶基因可以上调细胞内该基因的表达水平,同时促进维生素E含量的提高和/或组成的改变。依据主要作用的不同,五个关键酶大致可以分为两类——以提高维生素E含量为主要作用(包括HPPD和HPT)和以改变维生素E组成为主要作用(包括MPBQ MT、TC和γ-TMT)。在单基因转化的基础上,实验选择并设计了双基因转化策略,分别是组合γ-tmt+hpt和γ-tmt+mpbq mt,进一步验证了单基因转化评价的结论,并发现了双基因转化对同时提高维生素E的含量和改变维生素E组成有着十分显著的作用。基于此研究,以拟南芥为平台,建立了一整套可供评价比较的体系,为日后农业、经济作物的转基因工作提供有效的策略。2)通过基因工程方法调控生菜维生素E生物合成途径基于在拟南芥中的研究,选择拟南芥维生素E生物合成途径关键酶HPT和γ-TMT的基因,分别构建单基因转化和双基因共转化载体,组成性表达于生菜(Lactuca sativa L. var. logifolia)中,并获得含有目的基因的转基因株系。结果表明,单独转化hpt基因的株系可以提高维生素E生物合成途径的产物量,单独转化γ-tmt的株系可以改变维生素E的组成,使其中α-生育酚的比例大幅提高,双基因共转化株系较之非转基因对照(NC)和单基因转化株系,大幅度的提高了叶片维生素E的含量,最高达到了64.55μg/g FW,是NC株系叶片维生素E含量的9倍多,是hpt单基因转化最高表达株系的3倍多。由此得出结论,双基因共转化株系可以兼具提高维生素E总量以及改变组成的功能。另外值得注意的是提高下游关键酶γ-TMT的表达可以正向拉动生物合成,促进反应向下游进行,若同时提升上游关键酶的表达,可以明显提高最终产物量。本实验为改良蔬菜作物营养品质提供了一个有效的方法。3)基因工程调控维生素E生物合成途径对植物内源维生素C含量的影响以网络的角度,维生素E含量的提升可能会对转基因株系中抗坏血酸(维生素C)产生影响。本实验对过量单一表达维生素E生物合成途径关键酶的转基因株系叶片加以对比分析,发现内源维生素C的总量与维生素E的含量以及α-/γ-生育酚比例有负相关性。进一步的分析揭示在转基因株系中,参与Halliwell-Asada循环的关键酶基因(如apx、dhar和mdar)出现了表达上调,但维生素C生物合成途径相关基因并没有出现显著变化。这些发现初步揭示了脂溶性抗氧化物质维生素E与水溶性抗氧化物质维生素C之间通过Halliwell-Asada循环相互联系相互影响的关系,这对植物体内氧化还原内稳态的维持及植物生理活动有着重要的影响。本研究有助于进一步了解维生素E在植物生理中的作用,亦有助于为代谢工程提供实践的指导。4)改变拟南芥生育酚环化酶(TC)表达水平对维生素E生物合成的影响及其在逆境中的生理功能为研究维生素E在植物生理过程中的作用,通过基因工程方法调控关键酶TC的表达水平,构建了过量表达tc和RNAi抑制表达tc的转基因拟南芥株系,观察测量了这两种株系及NC株系在正常生理状态和逆境生理状态(盐逆境、干旱逆境)下的多种生理指标。结果表明,过量表达tc导致叶片γ-生育酚含量的显著提高,α-生育酚几乎不受影响；RNAi抑制tc的表达导致叶片生育酚含量的缺失,在正常生理状态下,虽不会致死,但植株出现了明显的生长延缓和矮弱现象,并导致维生素C含量的代偿性提高。施以不同程度逆境刺激后,与NC株系相比较,过量表达tc的株系的抗逆能力稍有增强。但是RNAi抑制tc表达的株系则出现了对逆境刺激的显著敏感性,一方面表现在体内应对逆境刺激能力的减弱,如脯氨酸和可溶性糖类积累水平降低；一方面表现在植株受到逆境伤害的程度增强,如脂类过氧化产物大量提高、细胞离子渗漏水平增强。这些发现初步证实了作为维持氧化还原内稳态的重要物质,维生素E对植物抵抗逆境有着重要的影响,协同维生素C共同发挥作用；并且发现γ-生育酚抗氧化的能力并不低于α-生育酚。更多还原

【Abstract】 Tocopherols, known as vitamin E, play paramount roles in the daily diet of both humans and animals as important lipid-soluble antioxidants. Tocopherols can be synthesized only in photoautotrophy organisms, including plants and other oxygenic, photosynthetic organisms, and hence human everyday needs of vitamin E are derived from plant materials or supplementary nutritional drugs. Recently, genes required for tocopherol biosynthetic pathway have been identified and cloned with the help of genomics-based approaches and molecular manipulation in the model organisms: Arabidopsis thaliana and Synechocystis sp. PCC 6803.Genes encoding five enzymes involved in tocopherol biosynthesis of Arabidopsis were cloned, and genetic manipulations were put forward with these genes in this study. Strategies were given rise to value the role of each individual gene in one plant species system, attempting to enhance the level of tocopherol content or convert the constitution of tocopherol. In addition, dual-gene transformation were performed in both Arabidopsis and lettuce (Lactuca sativa L. var. logifolia), succeeding in obtaining strains with elevated tocopherol contents andα-tocopherol composition. On the other hand, transgenic lines with target gene(s) overexpression or inhibition could be used to investigate tocopherol physiology in plant. The relationship of lipid-soluble antioxidant tocopherol and water-soluble antioxidant ascorbate, and function of tocopherol in plant defense were analyzed and discussed in this study.1) Systematic value of individual gene encoding enzyme involved in tocopherol biosynthetic pathway under Arabidopsis backgroundIn order to increase tocopherol content and to obtain desired tocopherol composition in plant, genetic manipulation of tocopherol biosynthetic pathway is an effective approach. In this study, five genes(HPPD, HPT, MPBQ MT, TC, andγ-TMT), encoding enzymes involved in tocopherol biosynthesis, were over-expressed in model plant Arabidopsis thaliana, either alone or in couple combinations (γ-TMT+HPT andγ-TMT+MPBQ MT), aiming to value and compare the functions of enzymes played in tocopherol biosynthetic pathway under the same genetic background. Our results suggested that, elevated expression level of biosynthetic pathway gene affected either total tocopherol content or composition, based on the position of he enzyme in tocopherol biosynthetic pathway. It can be divided into two groups because of the different functions -- the enzymes whose function are mainly on increasing total tocopherol contents (such as HPPD and HPT), and the enzymes effectively change the composition of tocopherols (such as MPBQ MT, TC andγ-TMT). In addition, dual genes transformation was predominant compared with single gene transformation strategies. Based on these studies, an appraisable system in sole plant was set up to value and compare the different roles of enzymes played in tocopherol biosynthetic pathway in term of tocopherol content and composition.2) Genetic engineering tocopherol biosynthetic pathway in lettuce (Lactuca sativa L. var. logifolia)In order to increase tocopherol content and to obtain desired tocopherol composition, genetic manipulation of tocopherol biosynthetic pathway can be employed as an effective approach. In this study, genes encoding Arabidopsis homogentisate phytyltransferase (HPT) andγ-tocopherol methyltransferase (γ-TMT) were constitutively over-expressed in lettuce(Lactuca sativa L. var. logifolia), alone or in couple combination. Our results suggested that, over-expression of hpt could increase total flux of tocopherol biosynthetic pathway, while over-expression ofγ-tmt could shift tocopherol composition in favor ofα-tocopherol. Transgenic lettuce lines expressing both hpt andγ-tmt produced significantly higher levels of tocopherol and vitamin E activity compared with non-transgenic control and transgenic lines harboring a single gene(hpt orγ-tmt). The best line produced 64.55μg/g FW total tocopherol content in leaves, which was over eight times of that in the non-transgenic control and more than twice the amount in the highest tocopherol-producing hpt single-gene transgenic line. We conclude that transgenic plants harboring both hpt andγ-tmt lead to increased tocopherol content and elevatedα-/γ-tocopherol ratio, which is more efficient than plants harboring only one of the two genes. The downstream enzymeγ-TMT plays important roles as a pulling force by improving the tocopherol composition, whereas the functioning of the upstream enzyme HPT is increased proportionally. Based on these studies, an effective approach is provided for improving the nutritional value of vegetable by increasing natural vitamin E content.3) Genetic engineering tocopherol biosynthetic pathway influence ascorbate level in transgenic linesWith the point of view of network, elevated tocopherol levels might have effect on other antioxidants in transgenic lines, especially ascorbic acid (AsA). It was found that metabolic engineering of tocopherol biosynthetic pathway affected endogenous AsA pool in leaves, which had negative correlation with tocopherol content and/orα-/γ-tocopherol ratio. Further study suggested that expression levels of genes encoding enzymes of Halliwell-Asada cycle were up-regulated, such as APX, DHAR and MDAR, while AsA biosynthetic pathway genes tested did not show remarkable changes. These findings provided hints on the relationship and regulation between lipid-soluble antioxidant tocopherol and water-soluble antioxidant AsA, which will help to perfect theory in plant physiology and give practical instruction for metabolic engineering.4) Overexpression and inhibition of tocopherol cyclase in Arabidopsis and its function in tocopherol biosynthesis and plant stress physiologyTocopherol functions have been studied most extensively in animal systems, and understanding of plant tocopherol functions pales by comparison. Tocopherols play important roles as antioxidants in many physiology processes. Transgenic lines with altered tocopherol levels and types will be important tools for physiology studies. In this study, expression level of gene encoding tocopherol cyclase was up- or down-regulated by transgenic methods. Changing in phenotype and physiology of transgenic and NC lines were investigated under normal, salt and drought stress state, respectively. Up-regulated of tc expression resulted in elevatingγ-tocopherol proportion, whileα-tocopherol content did not show marked increase compared with NC. RNAi inhibition lines succeeded in down-regulating tc expression level, and led to deficiency of total tocopherols. Decreased total tocopherol content was not lethal under normal growing state because of the complementary function of increased vitamin C level, but the strains were obviously weaker than NC. Tocopherol deficiency increased sensitivity of tc::RNAi transgenic lines toward all tested stress conditions, with altered proline or soluble sugar metabolism and increased lipid peroxidation and electrolyte leakage. However, tc overexpression lines showed an improved performance when challenged with salt or drought stress. These findings primarily confirm that vitamin E has important function in maintaining the balance of redox homeostasis and improving plant stress defense potential, with co-operation of vitamin C.γ-Tocopherol has similar antioxidant function asα-tocopherol.更多还原