DXS，Or和VHb基因在类胡萝卜素合成中的功能鉴定及其在水稻中的应用

Carotenoids are important nutrients and antioxidants for animals. Elevating carotenoid contents in foods has a great impact on human health. The possible factors limiting the overall carotenoid levels in “Golden Rice” could be (1) shortage of IPP precursor, (2) poor cooperation of different sets of carotenogenic enzymes and (3) lacking of a carotenoid-deposit mechanism in rice seed. This project aims to identify efficient DXS and Orange genes, which are involved in IPP formation and initiating chromoplast differentiation respectively, to solve the mentioned problems by establishing an Agrobacterium-based transformation system of rice callus. Furthermore, the elevatedβ-carotene will be converted to the value-added astaxanthin that can be esterized for sequestration by overexpressing β-carotene hydroxylase (CHYb) and ketolase (BKT). In addition, to increase the dissolved oxygen in rice cells, a critical factor for astaxanthin formation, the VHb gene will also be expressed. An polycistronic expression system based on 2A self-cleavage peptides will be adopted to ease vector construction and prove coordinate expression among the genes, which is important for the production of metabolites. It is expected that the rice seeds will accumulate much higher amounts of carotenoids including astaxanthin than ever made. Thus this project is of scientific and commercial significance.

类胡萝卜素是动物必须的营养物质和强抗氧化剂。提高粮食作物类胡萝卜素含量对人类健康具有深远意义。限制黄金米积累类胡萝卜素的关键因素可能是（1）缺乏IPP等前体；（2）低效率的内外源基因协作；（3）缺失类胡萝卜素储藏机制。本研究项目拟建立依赖于农杆菌介导的水稻愈伤快速筛选体系，筛选单子叶和双子叶植物中高效促进IPP合成的DXS基因和促进有色体分化与类胡萝卜素储藏的Orange基因。在此基础上增加β-胡萝卜素羟化酶（CHYb）和酮化酶（BKT）基因的表达使β-胡萝卜素转化为更高价值且利于高含量储藏的虾青素。同时为使氧依赖的BKT和CHYb发挥高效的催化功能拟进一步引入能增加细胞溶氧的VHb基因。多个关键酶基因将用2A序列连接来简化表达载体的构建和实现各基因的协同表达。本项目从增加前体、促进合成、降低产物降解等方面来探讨水稻种子中类胡萝卜素的合成和储存，具有重要的理论和现实意义。

水稻是最重要的粮食作物之一，但水稻种子中缺乏类胡萝卜素，长期以水稻为主食容易造成维生素A缺乏。“黄金米”将玉米的八氢番茄红素合酶（ZmPSY）基因和菠萝泛菌胡萝卜素去饱和酶（PaCrtI）基因导入水稻中表达，在水稻种子中合成并积累类胡萝卜素，用于解决维生素A缺乏的问题。1-脱氧-D-木酮糖-5-磷酸合酶（DXS）基因是类胡萝卜素合成前体异戊烯二磷酸（IPP）合成途径的关键基因，提高DXS基因的表达能提高IPP的含量。Orange（Or）基因能促进植物有色体的分化，从而为类胡萝卜素提供存储器官。本研究在“黄金米”研究的基础上，通过水稻愈伤鉴定了拟南芥DXS（AtDXS）、拟南芥Or（AtOr）、玉米DXS（ZmDXS）和玉米Or（ZmOr）基因对类胡萝卜素合成的促进作用，而且DXS和Or基因共同作用时类胡萝卜素含量高于Or基因,其中ZmDXS和ZmOr基因共同作用时类胡萝卜素含量最高，β-胡萝卜素达到127 μg/g湿重。之后，将ZmPSY、PaCrtI、ZmDXS、ZmOr以及莱茵衣藻胡萝卜素酮化酶（CrBKT）基因和雨生红球藻胡萝卜素羟化酶（HpCHY）基因在水稻种子特异性启动子控制下表达，希望得到高水平积累虾青素的转基因水稻。转基因水稻愈伤及幼苗分化过程中发现目的基因的渗漏表达使愈伤及植株叶片颜色发生改变，部分转基因植株分蘖变大，植株矮小。最终水稻种子中虾青素的含量为5 ng/g干重，但田间种植发现类胡萝卜素合成相关基因的在植株中渗漏表达严重影响水稻的正常生长，植株矮小且结实率低。