瓜叶菊MADS-box家族转录因子ScAG和ScAGL11在舌状花花斑形成中的功能解析

Pigment accumulation in the different parts of flower petals generates diverse color patterns, such as flower spots and stripe. It is a very important ornamental traits of ornamental plants and have great significant in plant evolutionary biology. However, the melocular mechanism for the diverse pigmentation pattern is still not clear. There is lots of cultivars with flower spots in Senecio cruentus which is a popular potted flower especially in spring festival in China. According to the previous comparative transcriptomic analysis, we find two genes, ScAG and ScAGL11, belonged to the MADS-box transcription factor family which are known to invovled in the flower development, showed dramatic different expression level between colored region and colorless region in petals. These two genes could repress the anthocyanin biosynthesis in Senecio cruentus and tobacco by transgene experiment. In this project, we will investigate whether ScAG and ScAGL11 can regulate the expression of anthocyanin biosynthetic genes by using yeast one hybrid, electrophoretic mobility shift assay and dual-luciferase reporter system. By using yeast one-hybrid screens we want to identify upstream transcription factors which can bind with promoter to activate ScAG and ScAGL11's expression. The high throught miRNA-Seq will be used to screen the specific miRNA that could regulate the expression of anthocyanin biosynthetic genes, ScAG, ScAGL11 and upstream regulatory genes. The expected results of this project will help clarify the functional roles of ScAG and ScAGL11 in the formation of flower spots in Senecio cruentus, and providing new insight into the molecular basis of anthocyanin metabolic regulation.

花瓣特定部位的花斑是决定花卉观赏品质的重要因素之一，且在植物进化上具有重要意义，迄今花斑形成的机理及色素基因定位表达机制尚不清楚，限制了观赏植物花斑定向育种。瓜叶菊是重要的盆栽花卉，具有很多花斑品种，是研究花斑形成机制的理想资源。我们前期发现了两个与花发育密切相关的C类MADS-box转录因子基因（ScAG和ScAGL11），在瓜叶菊斑色品种舌状花有色区和白色区显著差异表达，且均能抑制烟草和瓜叶菊中花青素的积累，是花青素代谢的负调控因子。本项目拟以ScAG和ScAGL11为切入点，创建RNAi株系，结合双荧光素酶、EMSA、Y2H研究其对花青素合成的调控机制；通过酵母文库筛选上游调控因子并分析其功能；利用高通量测序筛选特异miRNA，研究其对关键基因定位表达的调控作用。最终解析两基因在瓜叶菊花斑形成中的功能。上述研究对于高等植物花青素代谢调控机理及培育具花斑的园林植物新品种均具有重要意义。

瓜叶菊花色丰富，具有很多花斑品种。但决定其花斑形成的基因分离的相对较少，调控机制不清楚。我们在项目的支持下围绕瓜叶菊花斑形成的色素合成、细胞结构、关键基因及MADS-box基因家族ScAG和ScAGL11对花斑的调控机制进行了系统研究。通过扫描电镜发现斑部和非斑部的表皮细胞形态基本一致，有色区积累高含量矢车菊色素，斑区（白色）不积累花青素，而积累高含量的香叶木素和木犀草素等黄酮类物质，证明色素分支代谢决定瓜叶菊花斑形成。通过转录组分离两区域差异基因，发现转录因子ScAG、ScAGL11、ScMYBs、ScbHLH17、花青素代谢结构基因ScCHS2、ScF3H1、ScDFR3、ScANS及黄酮醇代谢结构基因ScFNS4和ScFLS5在斑色和非斑色区域差异表达。其中ScAG和ScAGL11在无色区高表达，而在有色区不表达。进一步研究发现ScAG和ScAGL11为细胞核定位蛋白，在烟草中过表达ScAG使过表达株系花青素含量明显下降。为了更快捷进行基因功能验证，我们建立了高效瓜叶菊叶片和花序的高效VIGS体系。并进行了所有差异转录因子基因和黄酮醇代谢结构基因的沉默研究，发现ScMYB3、ScMYB6和ScbHLH17参与瓜叶菊花青素积累的正向调控，且ScbHLH17与ScMYB2、ScMYB4互作，为瓜叶菊花青素代谢途径的关键MBW复合体。在花序中分别进行ScAG、ScAGL11及两者的共沉默，发现共沉默株系花序花斑白色区域完全消失，结构基因ScCHS2、ScDFR3、ScF3H1表达上调，而ScFNS4和ScFLS5的表达下调。酵母双杂交和双分子荧光互补试验表明ScAG与ScAGL11存在相互作用。通过酵母单杂交和双荧光素酶试验发现ScAG与ScAGL11通过分别抑制ScF3H1和ScDFR3的转录而负向调控瓜叶菊舌状花中花青素的生物合成。此外，斑色区和非斑色区的miRNA-mRNA联合测序结合qRT-PCR证明miR172-AP2模块、miR156-SPL模块可能参与瓜叶菊舌状花花斑形成的转录后调控。.本研究全面探索了瓜叶菊花斑形成机制，研究证明ScAG和ScAGL11互作通过直接影响下游色素分支调控基因的表达调控花斑形成。项目发表SCI论文4篇，均为第一标注。其中一区top期刊1篇，2区期刊1篇，3区论文2篇。培养研究生7名。