茉莉酸响应的AP2/ERF转录因子对菘蓝木脂素生物合成的调控作用及其分子机制研究

Lignans derivatives such as lariciresinol are main anti-viral active material basis with important medicinal value in Isatis indigotica. Limited resources and huge market demand will inevitably result in the supply and demand. Carrying out secondary metabolic engineering to improve their content is very important for the sustainable development of Isatis pharmaceutical industry. We predicted that the AP2/ERF transcription factors has a positive regulation role to lignan biosynthesis in Isatis indigotica based on the previously transcriptome and gene expression profile analysis upon MeJA. Four candidate family members were screened from 112 MeJA-responsed AP2/ERF transcription factors, suggesting that AP2/ERF transcription factor might regulate the lignan biosynthesis via monitor the gene expression level which involved in lignan or JAs pathway. This project intends to do the following studies as: ①. Phenotype analysis of the gene knock-out mutants via CRISPER/Cas9 gene edition tool to confirm the biologic function; ②. Using EMSA and ChIP to study the interaction between AP2/ERF transcription factors and DNA cis acting elements; ③. Sceening and functional characterization of the interacting protein with AP2/ERF transcription factors to construct the transcript regulatory networks by Y2H, Co-IP and BiFC etc. This work will clearly elucidate the regulation role and molecular mechanism of AP2/ERF transcription factors in lignan biosynthesis. It will also provide more adequate scientific basis for developing precise and efficient metabolic engineering to improve germplasm resources of Isatis indigotica by genetic manipulation of AP2 / ERF transcription factors.

落叶松脂素等木脂素类成分是菘蓝抗病毒活性物质基础，应用代谢工程技术提高其含量对于保障药材品质意义重大。通过转录组分析，我们预测AP2/ERF转录因子对菘蓝木脂素合成途径具有正调控作用，并从112条茉莉酸响应的AP2/ERF转录因子中筛选得到4个候选基因，提示菘蓝AP2/ERF转录因子可能通过响应信号分子，调控木脂素和茉莉酸合成途径关键基因的表达参与木脂素生物合成并起到重要作用。本项目拟以4个候选AP2/ERF转录因子为研究对象，采用CRISPR/Cas9构建基因敲除突变体以明确其生物学功能；采用EMSA和ChIP明确其与途径基因的相互作用及调控模式；采用Y2H、Co-IP和BiFC寻找和验证互作因子，解析转录调控网络，阐明AP2/ERF转录因子在菘蓝木脂素生物合成途径中的调控作用和分子机制，为后续开展以AP2/ERF转录因子为遗传操作靶标的精准代谢工程，改良菘蓝种质资源提供更充分科学依据

落叶松脂素等木脂素类成分是菘蓝抗病毒活性物质基础，应用代谢工程技术提高其含量对于保障药材品质意义重大。本项目基于菘蓝转录组数据库，通过典型相关分析方法筛选得到三个候选AP2/ERF转录因子（Ii008、Ii049、Ii099），对其进行克隆、鉴定、生物信息学分析和表达模式分析；构建基因过表达和抑制表达载体进行遗传转化，验证其生物学功能，其中Ii008和Ii049是正调控因子，Ii099是负调控因子；采用CRISPR/Cas9技术构建了Ii008基因敲除突变体并分析表型，共鉴定了27株基因编辑毛状根系，表明敲除Ii008对木脂素生物合成途径具有抑制作用；采用EMSA和Y1H明确Ii008与途径基因的相互作用及调控模式，结果表明Ii008直接结合途径基因启动子序列，具有直接的调控作用；采用水杨酸诱导处理方法明确了Ii049和Ii099对SA信号的响应，本研究通过对调控菘蓝木脂素生物合成途径的AP2/ERF转录因子的分离鉴定、功能解析及转基因研究，发现并识别高效促进落叶松脂素生物合成的关键调控因子，阐明了AP2/ERF转录因子在菘蓝木脂素生物合成途径中的调控作用和分子机制，为开展以AP2/ERF转录因子为遗传操作靶标的精准代谢工程，改良菘蓝种质资源提供更充分的科学依据。