REGULATION OF MAIZE PROVITAMIN A CAROTENOID BIOSYNTHESIS

类胡萝卜素来源于植物性食物，可在人体内转化为维生素A。维生素A不能从头合成，对生长发育至关重要。缺乏症表现为干眼症、失明、儿童疾病严重程度增加导致死亡率上升，以及艾滋病毒等病毒的母婴传播增加。与非维生素原A类胡萝卜素相比，玉米和小麦等粮食作物的胚乳中维生素A原含量较低；水稻胚乳完全缺乏任何种类的类胡萝卜素。全世界约有2.5亿儿童患有维生素A缺乏症，这与食用维生素A原类胡萝卜素含量低的食物有关；据估计，改善维生素A营养每年可消除约130万至250万人的死亡。维生素A干预计划已被证明只有适度的效果；缓解全球维生素A缺乏症的另一种方法是通过类胡萝卜素生物合成途径的代谢工程来提高食品中维生素A原类胡萝卜素的水平。为了通过代谢工程提高维生素原A类胡萝卜素的水平，我们必须了解类胡萝卜素的生物合成途径是如何被调节的，特别是在具有重要营养价值的种子胚乳中。我们认为维生素原A类胡萝卜素的相对积累是通过控制生物合成酶的转录水平来调节的；玉米胚乳中转录物水平的适当修饰（增强抑制）可导致维生素A原类胡萝卜素水平相对于其他非维生素A原类胡萝卜素水平升高。我们建议分离和研究额外的基因来验证我们的假设；我们将分离编码GGPPS（香叶基香叶基磷酸合酶）的cDNA(s)，这是该途径“上游”的关键酶，以及编码特异性影响维生素a原β -胡萝卜素相对积累的酶（LCYB， LCYE和HYDB）（番茄红素β -环化酶，番茄红素epsilon环化酶，β -胡萝卜素羟化酶）的基因。我们将重点关注cDNA的分离、转录、蛋白质和类胡萝卜素的分析，随后进行植物组织培养和转基因植物中转录水平的修饰实验。基因分离的新方法将涉及使用“颜色互补”，这是一种基于基因产物功能而不是核酸同源性的筛选方法。

Carotenoids, derived from plant food sources, are converted in humans to vitamin A. Vitamin A can not be synthesized de novo and it is essential for growth and development. Deficiencies manifest as xerophthalmia, blindness, increased mortality due to increased severity of childhood diseases, and increased maternal transmission of viruses such as HIV. Endosperms of food crops, such as maize and wheat, are low in pro- vitamin A as compared with non-provitamin A carotenoids; rice endosperms are completely deficient in carotenoids of any sort. About 250 million children worldwide suffer from Vitamin A deficiency that is associated with consumption of foods low in provitamin A carotenoids; it has been estimated that improved Vitamin A nutrition would eliminate approximately 1.3-2.5 million annual deaths. Vitamin A intervention programs have proven only moderately effective; an alternative approach to alleviating Vitamin A deficiency worldwide is to improve levels of provitamin A carotenoids in food stables through metabolic engineering of the carotenoid biosynthetic pathway. To metabolically engineer enhanced levels of provitamin A carotenoids, we must understand how the carotenoid biosynthetic pathway is regulated, particularly in the nutritionally important endosperm of the seed. We propose that the relative accumulation of provitamin A carotenoids is mediated by control of transcript levels for the biosynthetic enzymes; and that appropriate modification (enhancement of repression) of transcript levels in maize endosperm can lead to increased levels of provitamin A carotenoids relative to other non-provitamin A carotenoids. We propose to isolate and study of additional genes needed to test our hypothesis; we will isolate cDNA(s) encoding GGPPS (geranylgeranyl pyrophosphate synthase), a pivotal enzyme "upstream" of the pathway, and genes encoding enzymes that specifically affect the relative accumulation of the provitamin A betacarotene (LCYB, LCYE and HYDB) (lycopene betacyclase, lycopene epsilon cyclase, betacarotene hydroxylase). We will focus on cDNA isolation, transcript, protein and carotenoid analysis, followed by experiments leading to modification of transcript levels in plant tissue culture and in transgenic plants. Novel approaches for gene isolation will involve the use of "color complementation," a screening approach based on function of the gene product rather than nucleic acid homology.