转录因子IiWRKY34调控菘蓝优良品质形成的分子机制

The tetraploid I. indigotica with better yield and improved quality was induced from its natural diploid progenitor by our group. Compared with its diploid parents, its stress resistance and bioactivity are both significantly enhanced. However, the molecular basis of the significantly qualitative difference between tetraploid and diploid I. indigotica remains unclear. Our previous studies indicated that IiWRKY34 transcription factor was a key regulatory factor involved in the biosynthesis of lignans such as lariciresinol, which represent bioactive ingredients of Isatidis Radix. Besides, it also participated in root growth and stress resistance response. All of these suggested that IiWRKY34 probably take an important part in controlling the quality of I. indigotica, and the underlying regulatory mechanisms deserved further study. This project is to integrate the transcriptome and metabolome data of IiWRKY34 overexpression, IiWRKY34 depression and wild-type I. indigotica, to screen IiWRKY34 related genes, which involved in lignan biosynthesis, root development and stress resistance. Then, ChIP-Seq, EMSA, Y2H and CO-IP experiments will be carried out to confirm the regulation pattern and interaction network of IiWRKY34. Finally, IiWRKY34 will be transformed to obtain I. indigotica with improved bioactivity, stronger stress resistance and enhanced yield. This study will provide the theoretical basis and technical support for the sustainable supply of Isatidis Radix (the root of I. indigotica) with high quality.

四倍体菘蓝是本课题组通过染色体加倍培育的高产优良品系，与二倍体亲本相比，其抗逆能力及生物活性均显著增强。然而，导致两种倍性菘蓝品质差异的分子机制尚不够清晰。本课题组前期研究发现转录因子IiWRKY34是调控菘蓝活性成分落叶松脂素等木脂素生物合成的关键调控因子，且参与根系生长及抗逆响应，提示IiWRKY34可能是菘蓝优良品质形成的重要环节，其调控机制值得深入研究。本项目拟以过量表达、抑制表达IiWRKY34株系及野生型菘蓝为材料，进行转录组和代谢组整合分析，筛选IiWRKY34调控木脂素生物合成、根系发育及抗逆的相关基因；然后通过ChIP-Seq、EMSA、Y2H、CO-IP等明确IiWRKY34的调控模式和互作网络；最后通过IiWRKY34靶向调控，培育生物活性高、抗逆性强及高产的菘蓝优良品系，为保证优质板蓝根（菘蓝的根）药材的可持续供给提供理论基础和技术支撑。

四倍体菘蓝是本课题组培育的高产优良品系，抗逆能力及生物活性较其二倍体亲本显著增强。为探索两种倍性菘蓝品质差异的分子机制，本课题组前期对调控菘蓝活性成分落叶松脂素等木脂素生物合成的关键调控因子进行挖掘，提示IiWRKY34转录因子的表达可能是菘蓝优良品质形成的重要环节。为探究其调控机制，本项目以IiWRKY34过量表达、抑制表达及野生型菘蓝毛状根为研究材料，通过转录组和代谢组整合分析，构建“IiWRKY34-木脂素生物合成相关基因-木脂素含量”、“IiWRKY34-菘蓝根系生长发育相关基因”的相关性网络，筛选获得了IiWRKY34调控木脂素生物合成、根系发育及抗逆的相关靶基因Ii4CL3、IiNAC1、IiNAC3、IiNAC11和IiNAC18等；通过凝胶阻滞、酵母双杂实验等方法明确IiWRKY34的调控模式和互作网络，结果显示IiWRKY34可能通过调控以Ii4CL3为代表的木脂素生物合成途径基因的表达以调控木脂素类成分生物合成，通过调控根系发育相关转录因子IiNAC1的表达以调控菘蓝根系生长，而IiNAC1可能通过与IiNAC18结合来增强其调控功能；转基因拟南芥实验进一步明确了IiNAC1和IiNAC18促进植物根生长的生物学功能；通过IiWRKY34靶向调控，获得了生物活性高、抗逆性强及高产的菘蓝优良毛状根生物反应器。相关研究以项目负责人为通讯作者发表在Acta Pharmaceutica Sinica B等主流学术期刊；申请并获得国家发明专利授权1项。该研究为全面阐明四倍体菘蓝优良品质形成的分子机制奠定了基础，为推进菘蓝优良品系创制提供了科学依据。