F3'5'H调控马铃薯花青素合成流向的分子机理研究

Anthocyanins in potatoes have medical and health benefits. The pathway of anthocyanin biosynthesis has been basically clear. However, there are few studies reported on the regulation of flow direction of anthocyanin biosynthesis. In the early stage, we have fortunately obtained a mutant (red skin and red flesh) by the potato (purple skin and purple flesh) with in vitro mutagenesis technique. This project will identify the main pigment species and detect the difference in the expression of F3'5'H in tubers of purple potato and its red mutant, and the function of the gene will be analyzed. The above results will show that F3'5'H will be the main switch for anthocyanin biosynthesis flow. In order to explore the regulatory mechanism of F3'5'H expression, two tasks will be carried out. On the one hand, the differentially expressed genes and differential metabolites of the two materials will be analyzed by transcriptome and metabolome, respectively; the candidate genes will be obtained by their correlation analysis. On the other hand, the differential DNA sequences of the two materials will be sought by molecular marker technology, and its flanking sequences will be obtained by the hiTAIL-PCR technique, and the full length of the candidate genes will be acquired. The target genes will be obtained by comprehensive analysis of the candidate genes, and the target genes will be cloned and functionally verified. Finally, the relationship between F3'5'H and target genes will be analyze, and the molecular and regulatory mechanism of F3'5'H on anthocyanin biosynthesis flow direction will be elucidated.

马铃薯中的花青素具有医疗和保健作用。花青素生物合成的途径已基本清晰，但是在花青素合成流向调控方面缺乏研究。前期我们以紫皮紫肉的马铃薯为材料，采用离体诱变技术获得了红皮红肉突变体。本项目拟以紫色马铃薯及其红色突变体为材料，表征块茎中的主要色素种类，分析F3'5'H的表达量差异，进行该基因功能分析，明确F3'5'H是花青素合成流向转变的主要控制基因。为探索F3'5'H表达的调控机制，一方面通过转录组和代谢组分析两种材料的差异表达基因和差异代谢物，并对其进行了关联分析，获得候选基因；另一方面通过分子标记技术寻求两种材料的差异DNA序列，并利用hiTAIL-PCR技术获取差异DNA的侧翼序列，进而获取候选基因全长。对前期获得候选基因进行综合分析得到调控F3'5'H表达的靶标基因，对靶标基因进行克隆和功能验证。最后关联分析F3'5'H和靶标基因的关系，阐明F3'5'H调控花青素合成流向的分子机理。

以紫色马铃薯SD92和红色突变体SD140为实验材料，通过块茎色素的分离鉴定技术确定了它们的主要色素分别为矮牵牛色素和天竺葵色素。利用转录组与荧光定量技术确定了花青素合成流向由矮牵牛色素合成流向转变为天竺葵色素合成流向的关键基因为F3'5'H。然后利用转录组代谢组关联分析，并借助酵母单杂技术，挖掘了影响F3'5'H表达的调控因子MYBAS1、MADS、WD40重复蛋白和CBL相互作用的丝氨酸/苏氨酸蛋白激酶。与CBL相互作用的丝氨酸/苏氨酸蛋白激酶通过正向调控F3'5'H的表达来调节天竺葵色素苷的生物合成。MADS通过正向调控F3'5'H的表达来负向调控天竺葵色素苷的合成；WD40重复蛋白通过正向调控F3'5'H来负向调控天竺葵色素苷的合成，并组成LHW-bHLH与F3'5'H的启动子相互作用；MYBAS1通过负向调控F3'5'H的表达来调控花青素的合成。最后利用基因克隆技术和转基因技术，对调控因子MYBAS1的功能进行验证。本研究探讨了马铃薯块茎花青素合成流向的调控机制。这些结果丰富了花青素的代谢路径，为培育富含花青素的马铃薯品种提供了参考。