Ib-miR2111调控紫薯块根花色苷生物合成机制研究

Purple-fleshed sweet potato is a desirable resource for functional food development because of the abundant anthocyanins accumulated in its tuberous roots. The study on the mechanism of anthocyanin biosynthesis in tuberous roots of purple sweet potato is of great guiding significance to the breeding of new varieties of purple sweet potato with high value function. The expression regulation mediated by miRNA plays an important role in plant metabolism. Some studies have shown that miRNA is an important regulatory factor mediating anthocyanin biosynthesis in plants. However, the mechanism of miRNA regulating anthocyanin synthesis in the underground organs such as sweet potato roots has not been known clearly. In our primary study, Ib-miR2111 was discovered through transcriptome, small RNA and degradome sequencing analysis and was predicted to be related to anthocyanin synthesis. This project will use RLM-5' RACE and tobacco transient expression system to verify the target gene of Ib-miR2111. Its biological function will be elucidated through the over-expression and reduced-expression of Ib-miR2111 in sweet potato. Furthermore, the regulatory mechanism of Ib-miR2111 will be investigated via miRNA-mRNA co-expression network and gene co-expression network. This study will provide scientific theory and technical support for the cultivation of functional purple sweet potato varieties and the improvement of nutritional quality of other tuberous crops.

紫薯块根因富含花色苷而成为当前功能性食品开发的优质资源。紫薯块根花色苷合成机制的研究，对培育高值功能产品的紫薯新品种具有重大的指导意义。miRNA在植物多种生物代谢过程中起着重要的调控作用。已有研究表明miRNA是介导植物花色苷生物合成的一类重要的调节因子，但是有关miRNA调控甘薯块根等地下器官花色苷合成机制还未解析。Ib-miR2111是项目组前期通过转录组、小RNA和降解组测序分析挖掘到的可能与花色苷合成相关的小RNA。本项目将利用RLM-5′RACE和烟草瞬时表达体系来验证Ib-miR2111的靶基因；通过将Ib-miR2111在甘薯中过表达和减低表达来研究其生物学功能；并构建miRNA-mRNA共表达网络和基因共表达网络来解析Ib-miR2111介导甘薯块根花色苷合成的调控机制。本研究将为功能紫薯品种培育以及其他薯类作物营养品质改良提供科学理论及技术支撑。

紫薯块根因富含花色苷而成为当前功能性食品开发的优质资源。花色苷合成机制的研究，对培育高值功能产品的紫薯新品种具有指导意义。为了挖掘花色苷合成相关的miRNA和基因，本项目前期对紫心和白心甘薯进行了转录组、小RNA和降解组测序，挖掘到26个差异表达的miRNA和36个对应的靶基因。其中Ib-miR2111在紫薯中表达量显著高于白薯。通过降解组和烟草瞬时表达分析，鉴定出Ib-miR2111的靶基因为IbKFB。在拟南芥中过表达Ib-miR2111或者将AtKFB突变能够提高拟南芥植株的花色苷含量。构建Ib-miR2111和IbKFB的过表达载体和CRISPR/Cas9编辑载体，对甘薯进行遗传转化，共获得2个Ib-miR2111过表达转基因甘薯株系、28个CRISPR-miR2111基因编辑转基因甘薯株系、29个IbKFB过表达转基因甘薯株系和22个CRISPR-KFB基因编辑转基因甘薯株系。与非转基因甘薯相比，Ib-miR2111过表达和CRISPR-IbKFB转基因甘薯块根中花色苷含量增加；而CRISPR-miR2111和IbKFB过表达转基因甘薯块根中花色苷减少，表明Ib-miR2111通过抑制IbKFB的表达正调控花色苷的合成。进而对IbKFB的互作蛋白进行筛选，确定IbPAL、IbSAUR50、IbERF、IbGAPCp1为IbKFB的互作蛋白且可能参与花色苷生物合成。通过转录组测序，筛选出由于Ib-miR2111过表达或IbKFB基因编辑而引起的差异表达基因，并构建了基因共表达网络，初步揭示了Ib-miR2111及其靶基因介导甘薯花色苷生物合成调控机理。本研究将推动紫薯花色苷性状分子育种的进程，也为其他薯类作物营养品质改良提供科学理论及技术支撑。