不结球白菜ERF070转录因子参与调控其抗坏血酸生物合成的分子机制

The content of ascorbic acid (AsA) is an important nutritional quality indicator in the breeding of non-heading Chinese cabbage. However, due to its complicated factors such as environment, the research progress of AsA biosynthesis and regulation mechanism is slow. The preliminary study showed that the over-expression of BcERF070 in Arabidopsis could increase the content of AsA, while AsA content decreased after BcERF070 inhibited by VIGS technology in non-heading Chinese cabbage. In this project, transgenic technology was used to overexpress BcERF070 in low AsA content material and repress BcERF070 by RNAi in high AsA content material to obtain transgenic non-heading Chinese cabbage plants. The biology function of BcERF070 was identified by phenotypic analysis and VC analysis under different growth conditions. RNA-Seq technique was used to excavate the downstream genes regulated by BcERF070; The biological pathways of BcERF070 regulated in AsA biosynthesis were analyzed. The interaction genes of ERF070 were obtained by yeast two-hybrid technique, and verified by biochemical methods and others. At last we will clarify the molecular mechanism of ERF070 involved in regulation of ascorbic acid biosynthesis in non heading Chinese cabbage. Through this study, it will not only help to clarify the regulation mechanism of ascorbic acid biosynthesis in plant, but also will help to promote the breeding process of non - heading Chinese cabbage, which has important scientific significance and application prospect.

抗坏血酸（AsA）含量是不结球白菜品种选育的重要营养品质指标，由于其受环境等因素影响复杂，有关其合成调控机理的研究进展缓慢。前期研究表明，不结球白菜ERF070在拟南芥中异源过表达可使AsA含量升高；在不结球白菜中采用VIGS抑制后AsA含量下降。本项目拟采用转基因技术对低AsA含量材料进行BcERF070过量表达和高AsA含量材料RNAi抑制表达获得转基因植株，通过不同生长条件下表型和AsA含量分析明确BcERF070基因的生物学功能；应用转录组分析BcERF070调控的下游基因，解析BcERF070参与不结球白菜合成调控的生物学路径；通过酵母双杂交技术、生化验证等获得BcERF070互作基因并解析调控网络，从而阐明ERF070参与抗坏血酸合成的分子调控机制。通过本研究不仅有助于明晰植物抗坏血酸合成调控机制，而且有助于推动不结球白菜品质育种进程，具有重要的科学意义和应用前景。

抗坏血酸（AsA）含量是不结球白菜品种选育的重要营养品质指标，由于其受环境等因素影响复杂，有关其合成调控机理的研究进展缓慢。前期研究表明，不结球白菜ERF070在拟南芥中异源过表达可使AsA含量升高；在不结球白菜中采用VIGS抑制后AsA含量下降。本项目利用转基因技术获得过表达及CRISPR基因编辑植株，并结合植株表型和AsA含量分析明确了BcERF070对AsA的积累有正向促进作用的生物学功能。进一步利用酵母双杂交技术，获得其互作蛋白BcSRC2，通过双荧光素互补试验（LCI），双分子荧光互补技术（BiFC）以及pull down技术多角度验证了BcERF070和BcSRC2的蛋白相互作用。在不结球白菜中过表达BcSRC2，叶片中AsA含量显著高于野生型。RNA-seq分析表明，AsA-GSH路径的基因表达量在BcSRC2过表达植株和野生型植株存在显著差异，其中BcAPX4的表达量降低，BcDHAR1，BcDHAR2的表达量上升；APX和AAO等参与AsA降解的酶活性降低，DHAR等促进AsA再生的酶活性升高；BcSRC2的结合会抑制APX酶活性，BcERF070可使其抑制作用增强。这些结果揭示了BcERF070通过与BcSRC2互作，增强对APX的抑制，并参与AsA-GSH的代谢路径调控，从而提高AsA的含量。本研究还发现 BcERF070可增强下游BcBBX29基因的表达，提高植株AsA含量并延长植株营养生长时期。此外，ERF070的互作蛋白MLP328过表达也会降低植株中AsA含量。同时发现BcERF070与其互作蛋白在影响AsA含量的同时也会影响到开花时间，而有关AsA的合成调控与开花调控之间是如何介导的，还需进一步研究。这些研究成果为深入解析植物抗坏血酸合成调控机制，推动不结球白菜品质育种进程奠定了重要的理论基础。.在项目实施过程中，发表论文12篇；获得国家发明专利1件。获得神农中华农业科技一等奖1项，培养博士研究生3名，硕士研究生3名。