蓖麻Geranylgeranyl Reductase酶在维生素E高效合成途径中的作用研究

Vitamin E is a class of potent lipid soluble antioxidants that are essential active compund for human being and animals. Due to the nature of higher activity, the global demand for this limited resource of natural vitamin E which mainly comes from vegetable-oil industrial production is increasing by years. For vitamin E metabolic engineering, other than the significant achievement obtained for tocotrienol, only moderate progress was made for tocopherol. Late on, this was found to be due to the limited availability of substrate pool of PDP in those seeds. Regaring to the tocopherol biosynthesis, it has become apparent that the primary route of PDP synthesis in Arabidopsis, and likely all green seeds including canola and soybean, is via reduction of geranylgeranyl bound to chlorophyll rather by direct reduction of GGDP to form PDP; the non-green oilseeds such as castor bean and peach nut are only consist of direct reduction of GGDP to from PDP. Based on our previous findings that the limited ability of HPT overexpression to generate tocopherol enhancement in green seeds is likely due to relatively low substrate pools of PDP to drive tocopherol synthesis, and some of non-green seeds do accumulate relatively high levels of tocopherol in the absence of active chlorophyll synthesis. Therefore, we hypothesize that geranylgeranyl reductases from these non-green, tocopherol rich plant sources display a higher capacity for direct conversion of GGDP to PDP and may provide information for understanding the metabolic mechanism in non-green seeds and means for tocopherol enhancement in green oilseeds when the corresponding genes are coexpressed with HPT.

维生素E含生育酚和生育酚三烯两大类，是动植物体内重要抗氧化有机物质。主要油料作物籽粒内天然维生素E含量相对低，造成需大于供的矛盾。以提高发生范围更广的生育酚含量为目标的基因工程远不及以提高生育酚三烯为目标基因工程研究成功，主要因为可用于生育酚合成的前体PDP浓度相对较低。从PDP合成途径看，光合型种子（如油菜、大豆）以叶绿素分解途径为主，以Geranylgeranyl Reductase（GGR）酶催化GGDP生成PDP途径为辅；非光合型种子（如蓖麻、桃仁）只有GGDP到PDP的唯一途径。然而，非光合型蓖麻种子维生素E含量至少是大豆种子含量的1.7倍，说明蓖麻GGR酶应该具更强地生成PDP能力。本项目拟通过深入研究非光合型蓖麻籽内表达GGR酶的生化特性，在充分揭示其高效累积生育酚的代谢机制基础上，为进一步提高光合型油料籽粒内PDP含量，进而为显著增强主要油菜作物生产生育酚的能力提供新途径。

油料作物籽粒是天然维生素 E（生育酚）主要来源但含量较低，主要因为生育酚合成前体 PDP 浓度较低。从 PDP 合成途径看，光合型种子（如油菜、大豆）以叶绿素分解途径为主，以 Geranylgeranyl Reductase（GGR）酶催化 GGDP 生成 PDP途径为辅；非光合型种子（如蓖麻、桃仁）只有 GGDP 到 PDP 的唯一途径。然而蓖麻种子生育酚含量是大豆种子1.7 倍，表明蓖麻 GGR 酶可能具更强生成 PDP的能力。. 我们在拟南芥中超量表达蓖麻GGR酶并不能明显提高其种子生育酚含量，说明蓖麻GGR在拟南芥中并没有显著地增强GGDP向PDP转移；将蓖麻HPT（homogentisate phytyl transferase ，生育酚合成限速酶）在拟南芥进行了种子特异性超表达也不能明显增加生育酚含量；HPPD（4-Hydroxyphenylpyruvate dioxygenase）是生育酚合成另一前体尿黑酸合成关键酶，将蓖麻HPPD在拟南芥中超量表达可显著提高转基因种子生育酚含量，说明蓖麻HPPD活性强是蓖麻种子可高效合成生育酚的重要因素。. 在拟南芥中超量表达蓖麻GGR并没有显著提高PDP含量，可能是因为拟南芥具有光合型种子。已有体外证据表明叶绿素合成酶既可利用GGDP也能利用PDP合成叶绿素，但光合型种子内叶绿素合成酶是否可利用PDP合成叶绿素还不得而知。为解析这个问题，我们对叶绿素代谢与生育酚合成的关系进行了研究。结果发现，拟南芥叶片和发育种子叶绿素合成酶基因表达量与其生育酚合成能力间存在极显著负相关，说明叶绿素合成酶与生育酚合成关键酶HPT可相互竞争底物PDP，这限制叶绿素分解产物向生育酚合成方向进行高效转化，这也从另一个角度揭示了蓖麻种子因缺乏叶绿素从而由GGDP产生的PDP可全部用于生育酚的高效合成。. 以上研究，揭示了蓖麻种子因缺乏叶绿素合成酶基因这个生育酚合成的负调控因子而因此具备高效合成生育酚合成能力这一新机制。同时提出在油菜中可通过DNA编辑技术敲除油菜部分叶绿素合成酶基因，以及超量表达蓖麻HPPD或超量共表达HPPD+拟南芥HPT基因这两个途径进一步提高光合型油料籽粒生育酚含量。以上结果在Plant physiology上发表论文两篇。.