胡萝卜素羟化酶基因对胡萝卜肉质根类胡萝卜素合成调控的研究

Carrot (Daucus carota L.) is an important Apiaceae family root vegetables, abundant carotenoids accumulating in its edible fleshy root are essential nutrients for the human body, which are also the precursor of vitamin A and a good antioxidant and anticancer agent. In plants, carotenoids play an important role in photosynthesis, light protection, etc., and in the fleshy root of carrot, carotenoid is not only nutrients but also the main pigment for its various colour spectrum. Although the carotenoids biosynthesis genes in carrots have been identified, the mechanism of synthesis and degradation of carotenoids is not very intact. Our previous results demonstrated that the genes encoding carotene hydroxylase genes were strongly correlated with carotenoids biosynthesis during taproot development of different colour carrot. Based on the results of methods of morphological, physiological, anatomical, gene clone, promoter clone, gene expression profiles, yeast hybrid system, transgenic plants with over-expression and gene stable editing using CRISPR/Cas9 system, genome, transcriptome and proteome analysis, an extensive picture of the regulation mechanism of carotene hydroxylase genes involved in carotenoids biosynthesis of carrot taproot shall be attempted. On the basis of previous results, here, we mainly focus on the gene function of carotene hydroxylase genes and the regulation mechanism involved in carotenoids biosynthesis of carrot taproot. Some areas were highlighted below: anatomic characteristics of carrot taproot; distribution and content of carotenoid in carrot taproot; function of carotene hydroxylase genes in carotenoid biosynthesis. The function of carotene hydroxylase genes shall be studied by transgenic carrot plants. The results of the project shall not only help to ascertain the gene function of carotene hydroxylase genes involved in carotenoids biosynthesis of carrot taproot, but also provide a scientific basis for improvement of quality of carrot.

胡萝卜是伞形科重要的根菜类蔬菜作物，其可食用的肉质根中，能够积累丰富的类胡萝卜素。类胡萝卜素是人体所必需的营养物质，是维生素A合成的前体。目前胡萝卜中类胡萝卜素合成途径的相关基因已经被鉴定出来，但是类胡萝卜素合成机制仍待深入阐述。申请者前期研究结果发现胡萝卜素羟化酶与胡萝卜肉质根中类胡萝卜素合成密切相关。本项目在前期研究基础上以5种不同颜色和2种野生型胡萝卜为材料，围绕胡萝卜肉质根中胡萝卜素羟化酶基因功能，以及对类胡萝卜素合成调控开展研究。运用形态结构观察、类胡萝卜素测定、基因和启动子克隆、基因表达、酵母杂交、基因过量表达和基因敲除等技术，结合基因组、转录组和蛋白质组学分析，着重研究：胡萝卜肉质根形态结构特征；类胡萝卜素积累动态变化；胡萝卜素羟化酶基因与类胡萝卜素合成关系；转基因胡萝卜验证胡萝卜素羟化酶基因功能。研究结果将进一步丰富类胡萝卜素合成调控的分子机制，也为胡萝卜品质改良提供依据。

胡萝卜是一种重要的根菜类蔬菜作物。类胡萝卜素是一种天然色素，对植物的生长发育具有重要作用。胡萝卜肉质根中类胡萝卜素的组分和含量与肉质根的颜色密切相关。本项目建立了胡萝卜高效遗传转化和基因编辑技术体系。根据胡萝卜基因组数据，对胡萝卜胡萝卜素羟化酶基因、类胡萝卜素裂解双加氧酶基因和胡萝卜素双加氧酶基因等进行了检索和分析。利用生物信息学、转基因和CRISPR/Cas9基因靶向编辑等技术对胡萝卜DcBCH1、DcBCH2、DcCCD4和NCED进行了功能验证。结果发现胡萝卜肉质根中，DcBCH1的表达水平远高于DcBCH2。将DcBCH1和DcBCH2在胡萝卜中过表达，发现过表达DcBCH1基因的转基因胡萝卜肉质根中α-胡萝卜素和β-胡萝卜素含量均显著降低；过表达DcBCH2基因的转基因胡萝卜肉质根中的α-胡萝卜素含量降低，β-胡萝卜素含量变化不明显。利用CRISPR/Cas9基因编辑技术获得了DcBCH1敲除突变体，检测到突变体胡萝卜肉质根中α-胡萝卜素含量与对照胡萝卜相比略高，而β-胡萝卜素含量仍低于对照胡萝卜。此外，敲除DcBCH1的突变体胡萝卜肉质根中DcBCH2的相对表达水平显著升高。胡萝卜肉质根中，DcBCH1发挥了BCH酶的主要功能，DcBCH2对DcBCH1的功能具有补充或部分补偿的作用。过表达DcBCH1可通过调节类胡萝卜素的代谢来调控抗氧化能力，从而提高转基因植物的抗旱能力。将DcNCED2基因过表达载体中转入胡萝卜，验证DcNCED2基因的抗旱功能，并对DcNCED2基因在类胡萝卜素代谢中的分子调控机制进行探究。在类胡萝卜素积累量高胡萝卜材料中过表达DcCCD4，转基因胡萝卜肉质根中α-胡萝卜素和β-胡萝卜素的含量显著降低，而叶黄素含量变化不明显；在主要积累叶黄素的胡萝卜材料中过表达DcCCD4，结果显示转基因胡萝卜肉质根中叶黄素的含量略低于对照胡萝卜，DcECH基因的表达水平低于对照胡萝卜。利用CRISPR/Cas9基因编辑技术敲除胡萝卜材料DcCCD4，所获得的突变体肉质根中β-胡萝卜素和叶黄素的积累量增加，DcECH的表达水平显著高于对照胡萝卜。将DcCCD4进行原核表达和蛋白纯化，结果显示纯化的DcCCD4能以α-胡萝卜素和β-胡萝卜素为底物进行裂解。DcCCD4能通过裂解α-胡萝卜素和β-胡萝卜素影响胡萝卜肉质根中类胡萝卜素的积累。