miR156-SPL调控草莓果实成熟衰老过程中花色苷合成的分子机制研究

Strawberry fruits are rich in anthocyanins. Anthocyanins are not only important functional nutrients, but also an important sign of fruit ripening and senescence. Their synthesis and regulation have become research hotspot in recent years. Studies have shown that miRNAs play important regulatory roles in anthocyanin synthesis. However, the mechanism of miRNAs regulating anthocyanin synthesis during the ripening and senescence of strawberry fruits has not been reported. At the early stage of the project, miRNAs in strawberry fruits were identified, and it was found that miR156 was involved in the regulation of anthocyanin through the target gene SPLs, but the molecular mechanism of the regulation was still unclear. In this study, transgenic strawberry plants with miR156 functional inhibition and SPLs overexpression were obtained to clarify the regulation effect of miR156 on anthocyanin and SPL that plays a key role in anthocyanin synthesis. Combined with RNA-seq, EMSA, DLR and other research methods, the target genes regulated by SPL transcription factors were discovered and verified, and the molecular mechanism of miR156-SPL regulating anthocyanin synthesis was analyzed. The results can not only enrich the regulation mechanism of anthocyanin synthesis, but also provide a theoretical basis for the development of postharvest senescence regulation technology of strawberry, and the cultivation of strawberry varieties with strong anthocyanin and good storage resistance.

草莓果实含有丰富的花色苷，花色苷不仅是重要的功能性营养物质，也是果实成熟衰老的重要标志，其合成与调控是近年来的研究热点。研究表明，miRNAs在花色苷合成中发挥了重要的调控作用。但miRNAs调控草莓果实成熟衰老过程中花色苷合成的机理尚未见报道。项目前期分离鉴定了草莓果实中的miRNAs，发现miR156通过靶基因SPLs参与花色苷的调控，但调控的分子机制尚不清楚。本研究拟通过获得miR156功能抑制和SPLs超表达的转基因草莓植株，明确miR156对花色苷的调控作用及对花色苷合成起关键调控作用的SPL；结合RNA-seq、EMSA、DLR等研究手段挖掘和验证SPL转录因子调控的靶基因，解析miR156-SPL调控花色苷合成的分子机制。研究结果不仅能丰富果实花色苷合成的调控机理，也能为开发草莓采后成熟衰老调控技术、培育花色苷功能强化和耐贮性良好的草莓品种提供理论依据。

草莓中含有丰富的花色苷，果实成熟衰老过程中其总含量一直增加，花色苷不仅是重要的功能性营养物质，其含量变化也是草莓果实成熟衰老的重要标志之一。花色苷的合成与调控也一直是研究的热点。除了转录因子，近年来研究表明，miRNAs在花色苷合成中发挥了重要的调控作用。项目前期研究发现miR156在草莓果实成熟过程中差异表达，其靶基因为SPLs,可能参与花色苷合成的调控，但其调控机制尚不清除。本项目对不同时期草莓果实中花色苷含量进行了测定，发现草莓果实成熟过程中天竺葵素-3-O-葡萄糖苷含量显著增加，总花色苷含量增加，但总原花青素含量降低。筛选到了果实成熟过程中的差异表达基因，并对SPL家族基因表达模式进行分析，发现miR156的靶基因SPL12与花色苷含量变化趋势相反。采用烟草瞬时表达体系对SPL12蛋白进行亚细胞定位分析，结果表明，SPL12蛋白定位于细胞核。MYB10是草莓果实花色苷合成的正调控因子，双元荧光素酶结果表明SPL12转录因子可抑制MYB10启动子的活性。构建了miR156功能抑制及SPL12超表达载体并进行了瞬时转化验证，结果表明miR156功能抑制及SPL12超表达的草莓果实中花色苷合成减少。研究结果初步揭示了miR156-SPL12可通过抑制MYB10的活性从而抑制花色苷的生物合成。研究结果将丰富草莓果实花色苷合成的调控机理，也为开发草莓成熟衰老调控技术提供理论依据。