转录因子SmNAC2在丹酚酸生物合成过程中的作用及其机制研究

Salvianolic acid is the secondary metabolites of Salvia miltiorrhiza, and its biosynthesis is induced and regulated by many kinds of biotic and abiotic factors. NAC transcription factors are specifically identified in plants and they are widely involved in stress responses, growth /development, and metabolic regulation of plants. Our preliminary proteomc and bioinformatics studies showed that expression of NAC transcription factor 2 is significantly correlated with accumulation of Salvianolic acid. This project will confirm the role of SmNAC2 in Salvianolic acid biosynthesis and explore the underlying mechanism. Firstly, we will clarify the effect of SmNAC2 on expression of genes encoding salvianolic acid biosynthesis related enzymes and accumulation of salvianolic acid through overexpression and RNAi experiments. Secondly, we will screen out downstream genes of SmNAC2 using transgenic danshen plants through RNA-Seq and proteomic techniques. Finally, the detailed regulation sites and mechanism will be further elucidated by experiments such as electrophoretic mobility shift assays and chromatin immunoprecipitation. Completion of this project will clarify the molecular mechanism by which SmNAC2 affects salvianolic acid biosynthesis. Meanwhile, providing candidate gene for metabolic engineering of salvianolic acid biosynthesis.

丹酚酸是丹参的次生代谢产物，其生物合成途径受到多种生物及非生物因子的诱导调控。NAC是植物中特有的一类转录因子，广泛参与植物的胁迫应答、生长发育及代谢调控。课题组前期通过蛋白质组学及生物信息学研究发现，转录因子SmNAC2的表达与丹酚酸的累积显著相关。本项目拟深入研究SmNAC2在丹酚酸生物合成中作用及其分子调控机制：通过过表达和RNAi基因敲除实验，明确SmNAC2对丹酚酸合成关键酶基因及丹酚酸成分累积的影响；构建SmNAC2转基因丹参植株，运用RNA-Seq和蛋白质组学技术筛选SmNAC2调控的下游基因；进一步采用凝胶阻滞（EMSA）、染色质免疫共沉淀等技术验证SmNAC2的作用靶点。研究成果将阐明NAC类转录因子在丹酚酸生物合成中作用及其分子调控机制，为丹酚酸代谢工程研究提供靶向基因，对优良丹参资源开发进行有益探索。

丹酚酸是丹参中主要水溶性活性成分，对治疗心脑血管疾病有较好的疗效。NAC是植物中特有的一类转录因子，广泛参与植物的胁迫应答及次生代谢产物调控。NAC转录因子是否参与丹酚酸的生物合成调控，尚无报道。本课题对丹参进行了系统的蛋白质组学研究，共鉴定丹参蛋白3303个，UV-B辐射前后对比分析鉴定差异蛋白436个，进一步通过生物信息学及相关性分析发现转录因子NAC1与丹酚酸的含量变化成正相关；进而构建了NAC1过表达和RNAi转基因丹参发根体系，通过测定非转基因，NAC1过表达及RNAi发根中丹酚酸的含量变化确认了NAC1对丹酚酸生物合成的正向调控作用；进一步通过染色质免疫共沉淀-定量PCR(ChIP-PCR)及双荧光素酶报告（Dual-LUC）实验证实NAC1分别结合到丹酚酸合成限速酶苯丙氨酸氨裂解酶（PAL）PAL3和酪氨酸氨基转移酶（TAT）编码基因启动子区的CATGTG 和 CATGTC motifs来调控其基因表达和酶活力，从而促进丹酚酸的合成。研究成果以通讯作者或第一作者发表在The Plant Journal 一篇（IF=6.417,植物学领域一区TOP）、Frontiers in Plant Science (IF=5.753, 植物学领域一区TOP)、 Food &Function一篇(IF=5.396,农林领域一区TOP）。授权中国发明专利1 项。该研究明确了转录因子NAC1正向调控丹酚酸生物合成的作用，解析了内在调控分子机制，为丹酚酸代谢工程研究提供靶向基因，对优良丹参资源开发进行有益探索。