bHLH调控红豆杉紫衫醇生物合成的分子机理研究

Taxol, also called Paclitaxel, is a powerful and complex anticancer drug first isolated from the bark of Taxus brevifolia Pacific yew tree. However, taxol only constitutes 0.01-0.03% of the dry weight of the inner bark of the pacific yew tree. Intense research is being carried out to produce more taxol which would alleviate the supply problem. Total synthesis of this complex isoprenoid consist of multiple steps and are not economically feasible due to their low yield. Semi-synthesis production of taxol that consumes large amount of Taxus trees either cannot meet the requirement of the market. Cell culture production, especially induced by methyl jasmonate, can improve the total yields but the system is not stable. Taxol production by microorganism is another approach in recent years. Some fungi that produce taxol, or other related compounds, are being sought along with ways to enchance production. Transcription factors generally may be involed in intricate regulatory networks. These networks integrate the interactions of given genotypes with the environment and potentially control various branches of cell metabolism, activity and function. The bHLH family is one of the widest and most intensively studied families of transcription factors, which are defined by the basic helix-loop-helix (bHLH) signature domain. Arabidopsis MYC2 is a bHLH domain-containing transcription factor. Recent investigations have difined COI1/JAZs/MYC2 as a core JA signaling pathway module. MYC2 positively regulate sesquiterpnene biosynthesis. In our previous stduy, we carried out taxus RNA-seq work, and obtained seven bHLH gene sequences according to the taxus RNA-seq data. In this study, the role of bHLH transcription factor regulating taxol biosynthesis in taxus will be investigated by molecular biology methods. With the known sequences of bHLH, the over-expression vector and antisense RNA vector will be constructed to transform to taxus cell, respectively. Combined with both results of the expression patterns of the genes encoding the key enzymes related to taxol biosynthesis and the contents of taxol and baccatin III in the responsive cell lines, the molecular role of bHLH that regulate taxol biosynthesis will be conculded. To understand and employ bHLH 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.

转录因子参与萜类等多种化合物次生代谢过程，能够调控多个关键酶基因的表达。紫杉醇是非常重要的最为有效的抗癌药物之一，红豆杉生长缓慢，紫杉醇含量极低，供需矛盾日益激化。bHLH转录因子调控紫杉醇的生物合成的研究尚未见报道。本课题组前期研究发现了7个MeJA处理表达量升高的bHLH转录因子，且在T5OH等多个紫杉醇生物合成关键酶基因的启动子序列中发现bHLH转录因子结合位点，因此提出如下假说：bHLH极有可能对紫杉醇生物合成起到重要的调节作用。本研究拟利用红豆杉转录组数据信息，结合分子生物学技术，获得bHLH转录因子基因全长，并通过真核载体过表达或抑制表达bHLH，检测紫杉醇生物合成关键酶基因转录水平的表达变化，并测定相应转基因细胞系中紫杉醇的含量，深入研究bHLH对紫杉醇生物合成的调控作用，揭示bHLH调控紫杉醇合成的分子机理，得到紫杉醇产量得到提高的细胞系，为缓解紫杉醇供需矛盾提供理论基础。

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。转录因子能够调控萜类等多种化合物次生代谢过程，且有关bHLH转录因子调控紫杉醇生物合成的研究尚不多见。本项目利用项目组已有的红豆杉转录组数据信息，利用RT-PCR方法从南方红豆杉中分离克隆了一个bHLH转录因子家族基因——TcMYC，Genbank登录号为KC878013，并完成了该基因相关生物信息学分析、组织表达谱分析、茉莉酸甲酯诱导下的表达分析以及亚细胞定位研究，构建了真核表达载体，并进行了红豆杉细胞转化的工作。结果表明，TcMYC基因全长1,959bp，编码一个650个氨基酸残基的亲水蛋白，且结构域分析发现其N端包含有一个MYB/MYC类转录因子特有的保守域，下游C端有一个HLH结构域，与东北红豆杉（Taxus cuspidata）的MYC转录因子具有98%的一致性，亚细胞定位结果表明TcMYC转录因子定位于细胞核中。组织表达谱发现TcMYC基因在根、叶和木质部中表达量较高，而在嫩茎中表达量最低。TcMYC基因在茉莉酸甲酯处理后的红豆杉细胞中表达呈现略微下降趋势。目前研究正在进行TcMYC调控哪些紫杉醇生物合成关键酶基因的验证以及转化红豆杉细胞过表达TcMYC的工作，以期进一步揭示bHLH转录因子调控紫杉醇生物合成的分子机理，为缓解紫杉醇供需矛盾奠定理论基础。