NF-Y转录因子调控蓝莓果实成熟过程中花青苷积累的分子机制

Color development is crucial to quality formation during fruit ripening. The phenomenon of poor quality resulted from poor coloring is widespread in fruit production. Therefore, the research on fruit color development and regulation mechanism is crucial. Blueberry fruit is rich in anthocyanin, which is of great benefit to human health, however, the mechanisms of anthocyanin accumulation in blueberry fruits during ripening are mostly unknown. In this project, we investigate the regulation of anthocyanin biosynthesis by the VcNF-Y transcriptional factor in blueberry fruits during ripening based on transcriptomic and proteomic changes. By the comprehensive use of qRT-PCR, VIGS, EMSA, BiFC, Pull-Down and transgenic technology, combined with integrating omics, physiological quantification, histological procedures and tissue localization, the functions of VcNF-Y transcriptional factor family in anthocyanin synthesis during blueberry fruit ripening will be elucidated on the basis of existing research, as well as its interacting factors and downstream target genes will be profiled. The results of the study will further clarify the intrinsic molecular mechanisms of fruit color and quality development, reveal the signal intersection and regulation network during fruit maturing, and ultimately provide theoretical basis for directed molecular breeding and cultivation of fruit tree.

色泽发育是果实品质调控研究的重要内容。果树生产中普遍存在因着色不良导致的品质不佳等现象，开展果实色泽发育调控机理研究对提高果实品质至关重要。蓝莓果实富含花青素，具有极高的保健价值，但果实成熟过程中花青素积累调控的分子机制尚不清楚。本项目拟在课题组前期研究基础上，从组学水平研究NF-Y家族转录因子在蓝莓果实发育过程中的花青苷积累调控分子机制。项目将综合运用qRT-PCR、烟草脆裂病毒诱导的基因沉默技术、染色质免疫共沉淀、BiFC和Pull-Down及转基因等技术，结合串联组学分析、生理指标测定和显微观察等手段，筛选NF-Y转录因子的互作因子，鉴定其调控的下游靶基因，研究NF-Y转录因子在蓝莓果实成熟及花青苷积累调控中的作用，揭示果实成熟过程中信号交叉调控网络，阐明果实色泽发育与品质形成机理，为果树定向分子育种和优质高效栽培提供理论基础。

色泽发育是果实品质调控研究的重要内容。果树生产中普遍存在因着色不良导致的品质不佳等现象，开展果实色泽发育调控机理研究对提高果实品质至关重要。蓝莓果实富含花青素，具有极高的保健价值，但果实成熟过程中花青素积累调控的分子机制尚不清楚。在前期研究中，对蓝莓不同发育时期的果实进行了转录组测序，筛选出差异表达的VcNF-Y基因和花青苷合成通路酶基因，并拟对NF-Y参与蓝莓花青素合成调控的机制进行研究。研究发现，VcNF-Y家族基因随着果实成熟呈现下调趋势，在苹果果皮异源瞬时转化VcNF-Y基因部位果皮无变化，而注射花青苷合成通路酶基因，注射转化部位果皮出现不同程度的花青苷色块。其中，VcCHS、VcDFR2、VcDFR4过表达的部位花青苷色块颜色较深，VcCHI、VcANS、VcUFGT过表达的部位花青苷色块颜色较浅。说明转化体系可行，VcNF-Y家族基因在果实成熟过程中可能发挥负向调控作用，但未参与花青苷合成的调控。.因此，依托本项目，为探索蓝莓果实着色机理，我们进一步通过高通量测序技术，对北高丛蓝莓‘Legacy’进行转录组测序和sRNA测序，通过联合组学分析鉴定参与蓝莓果实成熟过程中的miRNA及其调控因子。通过一系列生信分析手段，结合生理数据，分析预测miRNA及其靶基因可能参与的果实成熟的各个过程及模式，并结合已发表降解组数据挖掘出了可能参与果实成熟和色素合成相关的miRNA及其靶基因，筛选出多对参与花青素和原花青素调控的基因对，并进行了功能验证，研究结果为果实着色的转录后调控提供了基础，拓展了果实成熟过程中色泽研究的调控通路。此外，蓝莓多为蓝色果实，粉色品种稀有，为进一步探究蓝莓着色差异机理，本项目选用了兔眼蓝莓中粉色果品种‘红粉佳人’和蓝色品种‘灿烂’果实不同发育期（大绿果、转色期果和蓝色果）进行转录组测序和代谢组联合测序分析，揭示了两个品种色素成分差异及着色差异机理。有意思的是，我们筛选出了调控蓝莓果实花青素合成过程中依赖光和不依赖光调控的关键MYB转录因子，为解决其他果树生产中因光照不足导致着色不良的问题提供了解决思路。