红豆杉AP2/ERF转录因子对紫杉醇生物合成的调控作用及其分子机制研究

Taxol, also named Paclitaxel, was firstly isolated from the bark of Taxus brevifolia Pacific yew tree as a powerful and complex anticancer drug. Taxol only constitutes 0.01-0.03% of the the dry weight of the Taxus bark, moreover, the Taxus tree grow very slowly. Therefore, a variety of methods are being carried out to produce more taxol to save more people's lives. . Transcription factors involved in terpenoid secondary metabolites, and AP2/ERF transcription factors involved in the regulation of the taxol biosynthesis. However, the further regulating molecular mechanisms have not been reported. The AP2/ERF transcription factors are widely present in the plant, which related a lot of physiological and biochemical reactions, such as disease resistance, stress tolerance, etc. Previous studies indicated that an AP2/ERF gene that isolated from Taxus cuspidata can bind the promoter of four genes which encoded Taxadiene Synthease, Taxane 5α-hydroxylase,Taxane 10β-hydroxylase, and Taxane 13α-hydroxylase of taxol biosynthesis. Besides, we also found the expression of 4 AP2/ERF genes was increased under MeJA induced from the taxus cell RNA-seq data. Among them, 2 AP2/ERF genes were cloned in our lab. According to our results, both of them expressed higher in roots and stems in Taxus marei, respectively. Further research should be carried out to study their regulating role in taxol biosynthesis.. In this study, the role of AP2/ERF transcription factor regulating taxol biosynthesis in taxus will be investigated by molecular biology methods and omics technology. According to the sequence information that obtained from our previous RNA-seq work and GenBank SRA database, 2-3 AP2/ERF will be cloned using RT-PCR and RACE methods. The CHIP-PCR assay will be performed to find the genes that encoding key enzymes of taxol biosynthesis. The over-expression vector and antisense RNA vector will be constructed to transform to taxus cell, respectively. With RNA-seq, itraq and GC-MS methods, the expression patterns of the genes and proteins that related to taxol biosynthesis and the contents of Taxanes will be investigated in the responsive cell lines. Combined all the described results, the molecular role of AP2/ERF that regulate taxol biosynthesis will be concluded. To understand and employ AP2/ERF regulating the taxol biosynthesis is helpful for meeting the taxol supply problem, and the taxus cell lines improved taxol production obviously is also very important for saving more lives of cancer patients.

AP2/ERF转录因子参与紫杉醇生物合成，能够调控紫杉醇生物合成途径中的4个关键酶基因，但有关其如何调控紫杉醇生物合成的分子机理的深入研究尚未见报道。本课题组前期研究发现4个MeJA处理表达量升高的AP2/ERF转录因子，并已克隆获得2个AP2/ERF基因cDNA全长，两者分别在红豆杉根和茎中表达量较高。本研究拟利用RT-PCR和RACE方法分离克隆2-3个与紫杉醇生物合成相关的红豆杉AP2/ERF基因，通过CHIP-PCR技术确定其调控紫杉醇生物合成的关键酶基因，并分别将AP2/ERF基因在红豆杉细胞系中过量表达和抑制表达，利用转录组学、蛋白质组学和代谢组学技术分析相应转基因细胞系中基因和蛋白的表达差异以及代谢组分的变化，明确AP2/ERF基因调控紫杉醇生物合成途径的关键步骤，全面系统揭示AP2/ERF调控紫杉醇生物合成的作用及其分子机制，最终为缓解紫杉醇供需矛盾提供新的理论基础。

紫杉醇是一种从红豆杉中提取的具有良好抗癌疗效的二萜类化合物，随着癌症患者日益增多导致紫杉醇需求量非常大，然而红豆杉却生长缓慢且紫杉醇含量极低，造成紫杉醇供需矛盾严重激化。转录因子可调控多个参与代谢途径基因的表达，因此，利用基因工程方法对转录因子基因进行操作，调控紫杉醇的合成产生具有巨大的应用前景和潜在价值。基于项目组前期转录组数据，本研究克隆获得了2个受到MeJA诱导参与调控紫杉醇生物合成相关的AP2基因cDNA全长，分别命名为TbAP2和TcAP2(TcERF)。针对这两个基因进行了生物信息学分析，进化树表明TbAP2和TcAP2属于两个亚类；，亚细胞定位表明两者编码蛋白均定位于细胞核，酵母单杂交结果显示它们分别与紫杉醇合成途径基因启动子中的DRE元件和GCC-box存在互作，对红豆杉中AP2/ERF基因家族进行了系统分析，表达特性分析表明TcAP2基因明显响应COR诱导，在处理0-14d期间表达量一直保持在5-10倍之间。基因转化结果表明，在拟南芥中超表达TcAP2对萜类骨架合成起到了抑制作用，抑制了细胞色素P450基因的表达，并增强了转基因植株的抗逆能力。在红豆杉细胞中过表达TcAP2基因抑制了TS等紫杉醇合成基因的表达，可见其对紫杉醇生物合成具有负调控作用。目前，相关转基因细胞系的转录组和代谢组检测工作正在进行中。此外，研究还建立了TbAP2和TcAP2最佳qRT-PCR实验体系，确定了红豆杉中COR、ABA、MeJA等处理下最适看家基因，完成包含紫杉醇（红豆杉）、丹参酮（丹参）以及佛波酯类化合物DPP（白角麒麟）等二萜类化合物上游合成途径中关键酶基因的挖掘工作，这些工作均为后续调控紫杉醇生物合成提供理论参考和奠定基础，也为后期研究扩展了新方向。综上，本研究基本达到了预期目标，揭示了TcAP2基因负调控紫杉醇合成的分子机理，为进一步阐明紫杉醇生物合成分子机制以获得稳定高产红豆杉细胞系提供理论依据。