REGULATION OF MAIZE PROVITAMIN A CAROTENOID BIOSYNTHESIS

类胡萝卜素来源于植物性食物，在人体内转化为维生素A。维生素A不能从头合成，它对生长和发育至关重要。缺陷表现为干眼症、失明、由于儿童疾病严重程度增加而导致的死亡率增加以及艾滋病毒等病毒的母婴传播增加。与非维生素A原的类胡萝卜素相比，粮食作物如玉米和小麦的胚乳的维生素A原含量低;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营养每年可减少约130万至250万人死亡。维生素A干预计划已被证明只有适度的有效性;一种替代方法，以减轻维生素A缺乏症的全球范围内是提高水平的维生素A原类胡萝卜素在食品稳定通过代谢工程的类胡萝卜素生物合成途径。为了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生物合成途径是如何调节的，特别是在营养重要的种子胚乳中。我们建议，维生素原A类胡萝卜素的相对积累介导的生物合成酶的转录水平的控制，并在玉米胚乳中的转录水平的适当修改（抑制的增强）可以导致维生素原A类胡萝卜素相对于其他非维生素原A类胡萝卜素的水平增加。我们建议分离和研究测试我们的假设所需的其他基因;我们将分离编码GGPPS（香叶基香叶基焦磷酸合酶）的cDNA，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（LCYB，LCYE和HYDB）（番茄红素β环化酶，番茄红素β环化酶，β胡萝卜素羟化酶）相对积累的酶的基因。我们将专注于cDNA的分离，转录，蛋白质和类胡萝卜素的分析，随后的实验导致在植物组织培养和转基因植物的转录水平的修改。基因分离的新方法将涉及“颜色互补”的使用，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.