辣椒果实中叶绿体与有色体调控辣椒红素积累的分子机制研究

Chromoplasts are the main organs that synthetize and store capsanthin. The ripening process of pepper fruits is typically accompanied by loss of chlorophyll and the synthesis of chromoplast. Previous studies focused on the study of chromoplasts in the mature chili fruit. Nevertheless, the global changes occurring during the differentiation of chromoplasts from chloroplasts have not been evaluated so far. In this study, 4 inbred lines with different colors were used as materials, chloroplasts and chromoplasts were separated at different stages of fruits development, characteristics of chloroplasts and chromoplasts were observed, and its chlorophyll and capsanthin contents were measured. The expression of proteins and genes involved in capsanthin accumulation was analyzed by Western blot and Real-time PCR, and the expression characteristics were identified. While iTRAQ technology was used to construct protein expression profiles of chromoplasts with high capsanthin content, and to analyze and screen the proteins involved in the cansanthin accumulation. On this basis, a regulatory network of protein interaction is established to explain the regulation mechanism capsanthin content. The results not only deepen understanding of the regulation mechanism of capsanthin synthesis, but also provide theoretical basis for the production regulation and genetic improvement of pepper fruit color and quality.

有色体是合成和储存辣椒红素的主要器官，辣椒果色从绿到红的变化是由于叶绿素的丧失和有色体的合成。前人的研究多集中在成熟期辣椒果实有色体的研究，然而在辣椒果实不同发育时期有色体分化的特征还未涉及。本项目以4个转色不同的自交系为材料，分别在果实发育的不同时期分离叶绿体与有色体，观察他们的形态结构、数量、空间排布等特征并测定叶绿素和辣椒红素的含量；采用Western blot及Real-time PCR技术对参与辣椒红素积累的蛋白和基因在翻译及转录水平的表达进行分析，明确这些蛋白和基因在不同亚细胞器间的表达特性；同时利用iTRAQ技术构建高辣椒红素有色体蛋白质表达谱，分析并筛选参与辣椒红素积累的蛋白质；在此基础上建立蛋白质相互作用调控网络来阐述调控辣椒红素含量的蛋白质间作用机制，研究结果不仅可以加深对辣椒果实辣椒红素合成调控机制的认识，而且可以为辣椒果实色泽品质的生产调控与遗传改良提供理论依据。

辣椒红素是从辣椒中提取的天然色素，其安全性已得到世界公认，辣椒红素可广泛用于食品、化工、化妆品和制药业中。目前辣椒红素的合成途径比较清晰，但合成过程的关键调控因子还不清楚。辣椒红素合成并储存于有色体中，因此研究辣椒红素含量积累的分子调控机制具有重要的意义。.本项目主要开展了5个方面的研究内容：①4个辣椒自交系不同着色时期辣椒果实叶绿素、类胡萝卜素和辣椒红素的含量测定；②4个自交系辣椒果实的叶绿体与有色体细胞学观察；③与辣椒果实着色相关的基因或蛋白qRT-PCR和Westernblot分析；④高辣椒红素品系的表达谱分析；⑤高辣椒红素品系的有色体提取与蛋白表达谱分析。.通过一系列的研究，取得了一些研究成果：辣椒红素的含量以辣椒果实为红色的品种最高；不同辣椒果实颜色其有色体分化的形态不同，青果期果实颜色为绿色的品系，有色体的形成由叶绿体发育而来，青果期为白色的的品系，其不含叶绿体；辣椒红素合成过程中的关键基因PSY、ZDS、CCS、CrtR-b、GGPS和pbsA均在辣椒红素含量高的品系中表达量高；通过对绿转红自交系辣椒果实不同着色时期的辣椒果实进行RNA-Seq分析发现，差异基因主要集中在生物调控、应激反应、信号、代谢过程、生长等的生物过程；富集在类胡萝卜素生物合成和淀粉、蔗糖代谢等信号通路，注释到AP2、MYB、MADS和ERF等类型的转录因子；对参与叶绿体与有色体发育且表达量差异明显的蛋白质构建互作网络，其中CHLP、CURT1C、FKBP16-3和APG1处于网络中心，表明其在辣椒红素积累过程中可能起到关键的调控作用。.本项目初步筛选的参与调控辣椒红素生物合成途径的转录因子或蛋白，为今后解析辣椒红素合成的调控机制奠定基础，同时也为培育高辣椒红素的专用型品种提供基础。