调控黄花龙胆玉米黄质环氧化酶基因表达的转录因子基因的克隆和功能分析

Carotenoids play a critical role in human nutrition and health. There have been many examples of unintented/unpredicted phenotypes and results in metabolic enginnering of carotenoids in higher plants. Typically this has resided from our lack of understanding of molecular regulation of carotenogenic gene expression and carotenoid formation in planta. Zeaxanthin epoxidase (ZEP) catalyzes the conversion of zeaxanthin to violaxanthin via antheraxanthin, and is the key enzyme responsible for the accumulation of antheraxanthin and violaxanthin in gentian (Gentiana lutea) flower petals. It has been demonstrated that the expression of gentian (G. lutea) zeaxanthin epoxidase (GlZEP) gene is strictly associated with carotenoid formation and chromoplast differentiation in gentian flowers and tomato fruits. In this project, the cDNAs encoding transcription factors for regulation of GlZEP gene expression will be isolated from cDNA library of gentian flower petals using Yeast One-Hybrid (Y1H) system, and characterized using transient expression system in tomato fruits by Agroinjection. The stable transgenic tomato plants over-expressing the putative transcription factor gene will be analyzed simultaneously for endogenous carotenogenic gene expression and carotenoid profile in different organs, flowers and fruits at different developmental stages. The genes coded for proteins that specifically interact with cloned transcription factor will be isolated and characterized from cDNA library of gentian flower petals using Yeast Two-Hybrid (Y2H) system and transient expression system in tomato fruits by Agroinjection. The anticipated outcomes of proposed project will provide insights into the molecular control mechanism of carotenogenic gene expression and carotenoid accumulation during the chromoplast differentiation in tomato fruits. The isolated and characterized transcription factor genes will be very useful molecular tools for the metabolic engineering of carotenoid biosynthesis and flower color modification in chromoplast-containing flowers and fruits.

类胡萝卜素对人类营养和健康具有重要价值。在应用遗传工程技术提高植物靶器官中某一特定类胡萝卜素含量时，存在很大的盲目性，这主要是由于目前人们对植物体内类胡萝卜素生物合成基因表达调控的分子机制以及类胡萝卜素积累的调控机制知之甚少。研究表明黄花龙胆玉米黄质环氧化酶(ZEP)基因表达与类胡萝卜素合成以及黄花龙胆花瓣和番茄果实有色体发育紧密相关。本项目拟用酵母单杂交和番茄果实瞬时表达技术从黄花龙胆花瓣中克隆和鉴定调控ZEP基因表达的转录因子基因；观测在番茄中稳定超表达转录因子对果实等器官内源类胡萝卜素基因表达和类胡萝卜素组分和含量的影响；用酵母双杂交等技术克隆和鉴定编码与转录因子相互作用的相关蛋白质基因；揭示他们在番茄果实有色体分化中对类胡萝卜素合成基因表达和类胡萝卜素积累的调控机制，为利用基因工程技术在含有有色体的花和果实等器官中生产类胡萝卜素以及改变植物花颜色的分子育种提供新的基因源和奠定基础。

类胡萝卜素对人类营养和健康具有重要价值。目前人们对植物体内类胡萝卜素生物合成基因表达调控的分子机制知之甚少。研究表明，与黄花龙胆玉米黄质环氧化酶基因(GlZEP)启动子相似，黄花龙胆八氢番茄红素脱氢酶、ζ-胡萝卜素脱氢酶、番茄红素β-环化酶和ε-环化酶以及胡萝卜素β-环羟化酶基因启动子驱动的报告基因在含有有色体的矮化番茄成熟果实中大量表达，而在含有叶绿体的未成熟的绿色果实中表达量极低。这些类胡萝卜素基因启动子都含有三个共有的顺式作用元件，即茉莉酸甲酯响应元件、乙烯响应元件和胚乳特异表达应答元件。这些共有的顺式作用元件可能代表负责调节这些基因共表达的转录因子的结合位点。克隆到与GlZEP启动子DNA序列中的乙烯响应元件相结合的矮化番茄RAP2.2转录因子基因以及与SlRAP2.2转录因子蛋白相互作用的转录因子SINAT2基因。.超表达SlRAP2.2转录因子基因下调番茄果实中GlZEP启动子驱动的报告基因表达和酶活性。与超表达一个SlRAP2.2基因相比，共表达SlRAP2.2和SlSINAT2转录因子基因的番茄果实中报告基因表达和酶活性略低,这表明这些转录因子负调控GlZEP基因启动子活性。. 黄花龙胆有两个不同花颜色（黄花或橙花）的变种。研究表明，橙花龙胆和黄花龙胆花瓣中的类胡萝卜素组分和含量以及黄酮和黄酮醇含量没有明显差异，但橙花龙胆花瓣中含有大量的天竺葵色素苷，而黄花龙胆花瓣检测不到任何花青素苷的积累。因此，橙花龙胆的橙色花瓣颜色主要是由新合成的具有红色调的天竺葵色素苷加在积累的黄色类胡萝卜素的背景中引起的，而橙花龙胆橙色花瓣中积累天竺葵色素苷主要是由于合成天竺葵色素苷的结构基因有较高的转录水平。另外，二氢黄酮醇还原酶（DFR）氨基酸序列的差异可能导致橙花龙胆DFR能够有效催化二氢堪非醇，最终导致在橙花龙胆花瓣中只积累天竺葵色素苷。. 克隆和鉴定的在含有有色体的花瓣和番茄果实中特异性表达的启动子以及橙花龙胆DFR基因将为利用基因工程技术改变植物花颜色和提高番茄果实中的类胡萝卜素和花青素苷等的分子育种提供新的基因源和奠定基础。