CsMYB60调控黄瓜黑刺类黄酮生物合成的分子机理

The spine color of cucumber is one of the important quality traits. Previous study showed that CsMYB60 was the best candidate gene for the B locus conditioning the pigmentation of black spines, but its biological function has not yet been validated and the mechanism of the coloration of black spines regulated by CsMYB60 still remains unclear. Our previous results indicated that the pigments in black spines are mainly flavonoids which play vital roles in resistance against cucumber root rot, and proved that CsMYB60 is a key factor for the pigmentation of black spines. In this project, we will clarify whether CsMYB60 directly regulates structure genes required for flavonoid biosynthesis by yeast one-hybrid and GUS transient expression system. The interacting proteins of CsMYB60 will be screened by yeast two-hybrid system, and verified with BiFC and CoIP methods, and then we will explore their biological functions with transient expression system in fruit spines. To verify the function of CsMYB60 and Cs4CL, these two genes will be transformed into the cucumber with white spine, then molecular identification, phenotypic analysis, component and quantitative analysis of flavonoids, transcriptomic analysis will be done in the transgenic plants. So, this project will elucidate the molecular mechanism of flavonoid biosynthesis controlled by CsMYB60 in cucumber black spines, which will provide theoretical basis for molecular breeding of quality and disease resistance in cucumber.

果刺颜色是黄瓜非常重要的外观品质性状。前人研究表明CsMYB60为黑刺基因B的最佳候选基因，但其生物学功能尚未确证，其调控黑刺形成的分子机理亦不清楚。本课题组前期工作明确了黑刺着色物质主要为类黄酮，发现其在黄瓜根腐病抗性中起重要作用，并证实了CsMYB60是调控黑刺颜色形成的关键因子。本项目拟利用酵母单杂交、GUS瞬时表达系统，明确该基因是否直接调控类黄酮合成途径中结构基因的表达；利用酵母双杂交筛选CsMYB60互作蛋白，用 BiFC及CoIP确证后，在果刺瞬时表达系统中验证其生物学功能，然后明确CsMYB60及其互作蛋白对类黄酮合成结构基因Cs4CL的调控模式；将CsMYB60和Cs4CL进行遗传转化，对转基因植株进行分子鉴定、表型分析、类黄酮测定以及转录组分析，进而解析CsMYB60调控黄瓜黑刺类黄酮积累的分子机理，为黄瓜品质及抗病分子育种提供理论依据。

果刺颜色是黄瓜的重要外观品质，但是黑刺中的色素成分及其生物合成调控机理却不清楚。在本项目中，我们利用代谢组学技术确定了黄瓜黑刺的主要色素物质是黄酮醇和原花青素，并且揭示了它们在黄瓜不同组织器官中的分布模式。转录组分析表明，苯丙烷代谢及类黄酮生物合成途径中的结构基因显著上调，这与黑刺色素成分鉴定结果一致。我们将转录组中所获得的SNP进行关联分析，把黑刺基因定位到了4号染色体末端，并获得候选基因CsMYB60；利用基因表达、瞬时转化以及遗传转化等技术，确定了CsMYB60就是黑刺基因；并且进一步发现白刺品系中CsMYB60表达量显著下调是由于Mutator转座子插入引起甲基化的结果。利用生物化学以及瞬时转化技术，我们明确了CsMYB60调控黄瓜黄酮醇和原花青素的分子机理：CsMYB60直接或间接激活CsbHLH42、CsMYC1、CsWD40和CsTATA-box的表达，然后与这4个蛋白相互作用形成复合体，结合到Cs4CL的启动子上激活其表达；同时，CsMYB60还可以和CsTATA-box相互作用形成复合体，结合到CsCHS的启动子上增强其表达；而且，CsMYB60能直接结合到CsFLS和CsLAR的启动子上激活它们的表达，进而促进黄酮醇和原花青素在黄瓜中的生物合成。黄酮醇和原花青素在人类慢性病预防以及植物抗病性等方面都发挥着重要作用，因此，本研究能为黄瓜品质育种以及抗病育种提供理论依据和靶标基因。