玉米黄素合成相关基因BCH参与调节菊花抗强光的分子机理解析

During the growth period, strong light is an important factor affecting plant growth. We had found that the β-carotene hydroxylase gene BCH cloned from Chrysanthemum morifolium was induced by strong light and played an important role in the regulation of zeaxanthin to protect the Chrysanthemum morifolium from injury caused by high light. However, the upstream regulation mechanism of Chrysanthemum morifolium BCH is still not very clear. We proposed to use the cut flower Chrysanthemum morifolium ‘Puma sunny’ as the acceptor material, to construct the overexpression vector and the microRNA interference vector, and to identify the function of the gene under strong light stress through the transgenic pathway; then, the wild and the transgenic strain are to be treated with strong light. Morphological, fluorescence parameters and zeaxanthin content are assessed, and the RNA-seq technology is used to analyze and screen the genes involved in the regulation. At the same time, the promoter region of BCH gene is cloned, and the photoresponsive elements sequences are obtained and screened by yeast one-hybrid technique. The upstream transcription factor is then analyzed for its molecular regulatory mechanism. All the results will well elucidate the function and resistance mechanism of Chrysanthemum morifolium BCH gene and provide theoretical basis for plant breeding and improvement of new resistance varieties.

生长期内强光是影响植物生长的重要因素, 课题组研究发现从菊花中克隆的β–胡萝卜素羟化酶基因BCH受强光诱导，参与调节生成玉米黄素，在保护菊花免受强光伤害方面发挥重要作用。然而，菊花BCH上游调控机制目前还不是很清楚。项目组拟以切花菊‘清露’为受体材料，构建超表达载体和microRNA干扰载体，通过转基因途径鉴定该基因在强光胁迫下的功能；然后，用强光处理野生株和转基因株，观测形态学、荧光参数和玉米黄素含量等方面的抗逆表现，并借助RNA-seq技术分析、筛选参与调控的基因，同时克隆BCH基因启动子区，获得光响应元件序列，应用酵母单杂交技术筛选上游转录因子，然后分析其分子调控机理。最终阐释菊花BCH基因的功能及抗强光的分子机制，为进一步选育和改良植物抗强光新品种奠定理论基础。

强光是影响切花菊产量和品质的重要逆境因子，抗强光胁迫相关基因β-胡萝卜素羟化酶基因CmBCHs的获得和抗强光的分子机制研究，将大大促进菊花及其他园艺作物抗强光新品种选育和改良。本项目构建了转化和非转化菊花‘神马’分别在正常光照和强光胁迫下的转录组数据库，获得4组差异表达基因。结果表明差异表达基因注释与生物过程类别中的次级代谢、微生物响应、非生物胁迫响应等相关；与分子功能类别中抗氧化、脂质代谢相关；与细胞组分类别中细胞壁、质体、呼吸链等相关。将构建的正义表达载体进行了拟南芥的转化，获得了菊花CmBCH1和CmBCH2转拟南芥株系各3个；以转CmBCH1和CmBCH2基因拟南芥和野生型（WT）为试验材料，在正常光照和强光处理下，进行形态、生物量、荧光参数、光合参数、色素含量、相关基因等指标的测定，比较不同株系在强光条件下的抗逆表现。结果表明CmBCH1和CmBCH2过量表达促进了类胡萝卜素合成相关基因表达量上调和光诱导基因的变化，使植株产生额外的叶绿素、玉米黄质和叶黄素，在强光下维持电子传递系统的完整性，降低了PSII反应中心的光抑制程度，从而更好地在强光条件下正常生长。扩增CmBCH1和CmBCH2基因上游启动子区，然后在Plant CARE上预测光响应相关的顺式作用元件。构建了菊花‘神马’叶片酵母单杂三框均一化cDNA文库。利用启动子全长序列构建诱饵载体，完成了bchpro-l Pro启动子筛库试验，最终在叶片cDNA 酵母文库中筛选并验证了钴离子跨膜转运蛋白ABCI12和脂肪酸羟化酶FAH与CmBCHs基因启动子互作。通过本项目的实施，进一步阐明了CmBCHs基因在抗强光过程的功能，为菊花及其他园艺作物抗强光遗传改良和高光效育种的定向设计提供更多的有效基因储备。

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