红皮石榴着色差异的机理研究

Pomegranate, with rich germplasm, is one of the superior and distinctive economic tree species in China. Various colorful resources of pomegranate are important material for anthocyanin biosynthetic regulation studies in fruit crops. Previous findings demonstrated that two red pomegranate cultivars ‘Hongbaoshi’ and ‘Moshiliu’ showed different anthocyanin gene expression profiles, but the molecular mechanism for these gene expression differences was not clearly illuminated. A series of methods, such as extensive targeted metabolomics, RNA-Seq, WGCNA, RT-PCR, qRT-PCR, yeast two-hybridization, bimolecular fluorescence complementation, and genetic transformation of tobacco will be carried out to explore the differential mechanism of coloration in red pomegranate. We will study the different accumulation profiles of flavonoids metabolites in two cultivars among different development phases, trying to reveals the physical basis of coloration differenation from metabolic levels. Also, we will study transcriptiome differences in two cultivars among different development phases, screening differential expression genes and flavonoids synthesis related genes. At the same time, based on WGCNA co-expression network analysis between differential expression genes and differential flavonoids metabolites, we will identify hub genes which are involved in anthocyanin biosynthesis in pomegranate, and verify their functions, trying to reveals the molecular basis of coloration differenation in two red pomegranate. The results not only expand the theoretical system of plants anthocyanin biosynthesis regulation mechanisms, but will be helpful for cultivation regulation and genetic improvement of pomegranate fruit color quality. Therefore, it is of great theoretical and practical significance to carry out the study.

石榴是我国优势特色经济林树种之一，种质资源丰富，不同色泽的石榴种质是研究植物花色苷转录调控的优异材料。前期研究发现，2个红皮石榴‘红宝石’和‘墨石榴’具有不同的花色苷基因表达模式，但调控这些基因差异表达的分子机制目前尚不清楚。本研究拟通过广靶代谢组学、RNA-Seq转录组学、WGCNA、RT-PCR、qRT-PCR、酵母双杂交、双分子荧光互补、烟草遗传转化等方法，研究两个品种着色期内果皮类黄酮代谢物的积累变化规律，从代谢水平揭示品种色泽差异的物质基础；研究两个品种着色期内转录组差异，筛选差异表达基因及类黄酮合成相关基因；基于差异基因与差异代谢物的WGCNA表达网络分析，鉴定参与石榴花色苷合成的关键基因，并对其进行功能验证，从分子水平揭示品种色泽差异的机理。研究结果不仅可拓展植物花色苷生物合成调控机理的理论体系，同时也有助于石榴色泽品质的栽培调控和遗传改良，具有重要的理论和现实意义

石榴我国优势特色经济林树种之一，在我国栽培历史悠久，遗传多样性丰富，具有多样的果实色泽。前期的研究表明，2个红石榴品种‘红宝石’和‘墨石榴’可能具有不同的色泽调控模式。本项目以红皮品种‘红宝石’、紫皮品种‘墨石榴’为材料，通过代谢组学、转录组学、瞬时表达、拟南芥遗传转化等方法，研究了两个品种色泽差异的代谢及分子基础。总结了着色期内主要花色苷组分及含量的积累变化规律，发现6种花色苷Cy3G、Dp3G、Pg3G、Cy3,5dG、Dp3,5dG和Pg3,5dG的含量差异是造成两个品种色泽差异的主要原因；‘红宝石’中Pg花色苷含量随发育期逐渐下降，‘墨石榴’中Cy含量逐渐下降，Dp含量逐渐上升。在发育前期，果实中以双花色苷为主，成熟时以单花色苷为主。开展了着色期内转录组分析，筛选出两个品种着色期内以及同一着色阶段的不同对比组的差异基因，通过KEGG富集分析，鉴定出参与石榴花色苷合成的结构基因21个，包括PAL、CHS、CHI、F3H、DFR、ANS、UGT、F3'H、F3'5'H、FLS、LAR、ANR，其中，ANR基因在‘墨石榴’中的低表达是两个品种着色差异的分子基础；此外，14个MYB、6个bHLH、3个WD40、2个bZIP等转录调控基因可能参与了石榴花色苷的生物合成。选择了2个MYB和2个bZIP基因进行了功能研究。分别克隆了PgMYB11、PgMYB111、PgbZIP16、PgbZIP34等四个转录调控因子，GFP亚细胞定位分析发现它们都位于细胞核；烟草瞬时表达结果表明四个转录因子可促进烟草叶片花色苷的积累；转PgMYB11和PgbZIP16的阳性拟南芥植株中，花色苷合成相关基因与PgMYB11和PgbZIP16的表达量比对照有显著提高，且GUS染色结果发现PgMYB11和PgbZIP16具有组织转录活性，因而确认了PgMYB11和PgbZIP16对石榴花色苷有正向调控作用。这些研究结果对石榴果实外观色泽品质的网络调控研究和相关育种研究具有重要的理论与实践意义。