CCD4a和CCD4b基因在甜橙果实色泽形成中的功能解析

Fruit color is an important constituent of fruit quality. Carotenoid cleavage dioxygenases (CCDs) exert their influences on fruit coloration by cleavage of specific carotenoids. Our previous study found that a red-flesh sweet orange Cara Cara accumulated more abundant lycopene compared to its wild type. Recent Solexa sequencing revealed that two CCD4 genes (CCD4a and CCD4b) showed significant difference in transcriptional expression between the red-flesh mutant and wild type and they exhibited opposite expression patterns, suggesting that these two CCD4 genes may play different roles in carotenoid accumulation in citrus fruits. In this study, we will measure the expression levels of all CCD genes predicted in the citrus genome and examine their possible relationships with carotenoid accumulation in sweet orange fruits. As a priority of this project, the biochemical function of CCD4a and CCD4b in carotenoid accumulation will be investigated by enzymatic assays using bacterially expressed proteins in vitro or in vivo by transfecting E. coli strains accumulating carotenoid substrates with the CCD4 genes. The role of these two genes will also be investigated in planta by over-expression and RNAi silencing of the CCD4 genes in Hongkong kumquat (Fortunella hindsii Swingle), a citrus relative with a very short juvenile period. Expected results from this work will help clarify the functional roles of the CCD4 genes in fruit color-development of sweet oranges, and consequently provide insight into the molecular basis of citrus fruit coloration valuable for genetic improvement in future breeding programs.

色泽是柑橘果实品质的重要组成部分，类胡萝卜素裂解双加氧酶（CCD）通过降解特定类胡萝卜素成为影响果实色泽的重要酶。本项目前期研究发现一个甜橙红肉突变体红肉脐橙与野生型相比富集了大量番茄红素；近期Solexa深度测序发现两个CCD4基因（CCD4a和CCD4b）在红肉突变体和野生型中极显著差异表达，且表达模式相反，暗示这两个基因与甜橙类胡萝卜素成分变异有关，且两者编码的酶的作用底物可能不一样。本研究拟研究甜橙全基因组CCD基因与果实类胡萝卜素形成的关系；采用蛋白原核表达与体外孵育、类胡萝卜素工程菌体系确定CCD4a和 CCD4b的作用底物；在短童期的山金柑中进行超表达和RNAi，重点验证CCD4a和CCD4b基因的功能，并评价基因操作对果实色泽和其他品质的影响。研究结果将明确CCD4基因在甜橙类胡萝卜素形成中的功能，为柑橘果实色泽分子改良和调控奠定基础。

色泽是柑橘果实品质的重要组成部分，然而调控果实色泽的关键基因尚不清楚。课题组前期研究发现类胡萝卜素裂解双加氧酶基因4（CCD4）可能参与了柑橘果皮色泽的形成。基于此，本项目克隆并分析了橘橙柚等三十余个柑橘品种的CCD4亚家族各成员基因（CCD4a、CCD4b、CCD4c、CCD4d、CCD4e），发现CCD4基因存在大量的SNP、缺失和移码变异。通过分析CCD4亚家族各成员在橘橙柚中不同组织的表达情况，发现CCD4b特异的在红色或橙红色果皮中表达。利用纽荷尔不同发育时期的果皮和果皮颜色不同的柑橘品种进行基因表达与β柠乌素（决定果皮颜色的关键物质）的相关性分析，结果表明CCD4b与β柠乌素相关性很高。同时测定红橘（果皮红色）和枳壳（果皮黄色）杂交群体中不同颜色果皮的β柠乌素含量及其CCD4b基因的表达，验证了CCD4b表达量和β柠乌素的正相关性。我们平行采用枳壳和红橘的杂交群体检测了杂交后代果皮β柠乌素含量，选择了黄色和红色两类极端表型，构建两个DNA混合池，采用BSA-seq技术分析影响果皮红黄表型的区域，进一步采用CAPS标记进行精细定位，锁定到2Mb区域，这个区域包括了CCD4b基因，且仅该基因参与类胡萝卜素代谢，这进一步说明CCD4b是调控柑橘果皮色泽的关键基因。CCD4b基因序列分析发现，CCD4b启动子区域存在MITE（微小反向重复转座元件）的插入事件，且不同品种中的MITE插入的数量和种类存在差异。其中一个MITE（60bp）在红皮柑橘的CCD4b基因中都存在，而在黄皮柑橘中则都不存在。综合以上研究结果，CCD4b的表达高低是决定红黄果的关键原因，而其启动子区域的MITE插入很可能是导致表达量差异的原因。该研究锁定了决定柑橘果皮颜色的关键基因并发现了导致颜色差异的原因，为柑橘果实色泽品质育种提供了优良的供体基因，具有重要的理论和实践意义。