REGULATION OF MAIZE PROVITAMIN A CAROTENOID BIOSYNTHESIS

玉米维生素原 A 类胡萝卜素生物合成的调控

• 批准号: 6347535
• 负责人: ELEANORE T WURTZEL
• 金额: $ 6.38万
• 依托单位: HERBERT H. LEHMAN COLLEGE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6347535
• 关键词: carotenoids; complementary DNA; corn; developmental genetics; electron microscopy; enzyme activity; gene complementation; gene expression; gene mutation; genetic library; genetic manipulation; genetic mapping; genetic transcription; genetically modified plants; geranyl compound; molecular cloning; northern blottings; nucleic acid sequence; plant genetics; polymerase chain reaction; pyrophosphates; retinoids; southern blotting; transposon /insertion element; vitamin biosynthesis

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.

类胡萝卜素来源于植物食物，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。缺陷表现为干眼症、失明、由于儿童疾病的严重性增加而导致的死亡率增加，以及艾滋病毒等病毒的产妇传播增加。与非维生素A原类胡萝卜素相比，玉米和小麦等粮食作物的胚乳中维生素A原含量较低；水稻胚乳中任何种类的胡萝卜素都完全缺乏。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营养将每年减少约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效果；缓解全球维生素A缺乏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原胡萝卜素的水平。为了提高维生素A原类胡萝卜素的代谢水平，我们必须了解类胡萝卜素的生物合成途径是如何调节的，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A原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；玉米胚乳中转录水平的适当调节(增强抑制)可以导致相对于其他非维生素A原类胡萝卜素的维生素A原胡萝卜素水平的增加。我们建议分离和研究验证我们的假设所需的其他基因；我们将分离编码GGPPS(香叶素焦磷酸合成酶)的基因(S)，GGPPS是该途径的上游关键酶，以及编码特定影响维生素A原β胡萝卜素相对积累的酶(LCYB、LCYE和HYDB)的基因(番茄红素β环酶、番茄红素表观环化酶、β胡萝卜素羟基化酶)。我们将集中于cDNA分离、转录物、蛋白质和类胡萝卜素的分析，然后是在植物组织培养和转基因植物中导致转录水平改变的实验。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的功能而不是核酸同源性的筛选方法。