响应内生菌深绿木霉促进丹参酮合成的关键转录因子及高含量毛状根体系的构建策略

Medicinal value of tanshinone compounds have been found constantly and clinical demand of tanshinones rises sharply. However, traditional cultivation of medicinal plants, chemical synthesis and cell and tissue culture production cannot meet pharmaceutical industry needs of tanshinone compounds.In our previous studies, we found that Trichoderma atroviride D16 could increase the content of tanshinone significantly, and three key transcription factors in response to D16 were obtained by combining of transcriptome and metabolomics. Firstly, key transcription factors in response to D16 were cloned and key transcription factor- knockout hairy root was established to clarify its function. Secondly, regulation mechanism of key transcription factor inducing tanshinones accumulation was studied through chromatin immunoprecipitation sequence(ChIP- Seq), yeast one- hybrid, electrophoretic mobility shift assay(EMSA) and other technologies. Thirdly, to form a new system of high-yielding tanshinone hairy root, the overexpressed hairy root system of key transcription factor were constructed and extraction of D16 mycelia was optimized as an additive. On one hand, complete of the project can reveal the scientific mechanism of D16 regulating tanshinoes accumulation. On the one hand, it will provide useful practical experience for the efficient production of plant secondary metabolites.

由于丹参酮类化合物的药用价值被不断发现，临床需求量骤增，而传统栽培药材、化学合成以及细胞和组织培养生产丹参酮均不能满足制药工业的需要。我们前期研究发现丹参内生真菌Trichoderma atroviride D16能大幅提高丹参酮含量，结合转录组和代谢组学筛选得到响应D16调控丹参酮的3个关键转录因子。本项目计划：（1）对筛选到的响应内生真菌诱导的关键转录因子进行克隆，构建基因敲除毛状根，明确其作用；（2）通过荧光素酶测定、染色质免疫沉淀和转录因子微孔板芯片检测等技术对关键转录因子的作用机制进一步分析，明确其作用模式和作用靶点，阐明调控机制；（3）构建高效的关键转录因子过表达毛状根体系，同时优化D16活性部位的提取工艺，制备成添加剂，形成新的高产丹参酮毛状根体系。本项目的完成，一方面将揭示内生菌D16调控丹参代谢的科学内涵；一方面将为生物技术定向高效生产植物次生代谢产物提供有益的实践经验。

随着丹参酮类化合物的药用价值被不断挖掘，临床需求量巨大，而传统方法均不能满足制药工业的需要。为此，我们前期筛选出一株内生真菌Trichoderma atroviride D16能大幅提高丹参酮含量。按照项目研究目标，（1）首先对D16多糖部分（Polysaccharides Fraction, PSF）诱导的丹参毛状根和对照组丹参毛状根进行转录组测序，筛选得到2条ERF转录因子SmERF1b和SmERF1-like。利用烟草瞬时转化技术明确SmERF1b和SmERF1-like转录因子均定位于细胞核中。挖掘SmERF1b和SmERF1-like在丹参转录组数据中根、茎、叶的表达分布情况，并采用qRT-PCR进行验证，均显示SmERF1b和SmERF1-like在根中表达量最高，在茎中表达量次之，在叶中几乎不表达。采用qRT-PCR测定D16 PSF诱导丹参毛状根中的转录因子表达量，结果显示SmERF1b和SmERF1-like的表达量均随着D16 PSF的诱导时间而显著升高。（2）分别构建SmERF1b和SmERF1-like敲除毛状根体系，进行丹参酮含量测定和表型观测。结果显示SmERF1b敲除后的丹参酮含量显著下降，而生物量无显著变化；SmERF1-like敲除毛状根的生长受阻。酵母单杂实验表明SmERF1b可与丹参酮生物合成途径SmKSL1启动子的GCC-box结合，而SmERF1-like可同时与丹参酮生物合成途径酶基因SmIDI1和SmKSL1启动子的GCC-box结合；凝胶迁移率实验进一步确定了SmERF1b和SmERF1-like与丹参酮生物合成途径酶基因的结合情况，表明SmERF1b可以直接调控丹参酮生物合成途径的SmKSL1，而SmERF1-like可同时参与调控SmIDI1和SmKSL1。（3）进一步构建了SmERF1b和SmERF1-like过表达毛状根，为毛状根的高丹参酮生产体系建立提供技术支持。此外，明确了D16作为固体生物菌肥的作用效果，同时确定其最佳施用量为15g D16生物菌肥/棵。大田试验也表明固体菌肥对丹参生长和丹参酮的促进作用远远高于已研究的丹参菌肥。本项目探讨了内生菌D16诱导子PSF促进丹参酮合成的作用机制，为毛状根高产丹参酮体系建立以及栽培丹参品质提升提供重要的研究基础。