乌头剧毒特性关键BAHD酰基转移酶基因的鉴定与功能研究

The acute toxicity of Aconitum carmichaeli，one of the main traditional herbal medicines, severely limits its exploitation and utilization in clinical treatment. Then, it is of great importance to elucidate the toxicity-formation mechanism of A. carmichaeli for solving the toxicity of A. carmichaeli. Extensive investigations indicate that the high toxicity closely relates with the ester groups on the skeleton of diterpenoid alkaloids(DAs), which are the primary secondary metabolites of A.carmichaeli. Furthermore, BAHD acyltransferases are the last catalytic enzymes that control the biosynthesis of ester groups. Based on the collection of germplasm resources of Aconitum spp in Yunnan province, we obtained the A. carmichaeli with the principal components rich in aster groups and acute toxicity and A. weixiense with the principal components absent of aster groups and nontoxicity by the isolation of active substances and their activity evaluation. Accordingly, comparative transcriptome analysis will be adopted to selected out the candidate BAHD genes of A. carmichaeli based on the gene differential expression, expression traits of tissue specificity and enrichment pathway analysis by the sequence, together with the functional characteristics of the branches of the constructed phylogenetic tree from BAHD genes for the two Aconitum plants. Combined with the temporal and spatial expression of candidate BAHD genes from different tissues in various developmental stages for A. carmichaeli, and the change of ratio between the contents of primary DAs with aster groups and relevant DAs without ester groups, key BAHD candidate genes would be identified. The target BAHD proteins will be obtained by gene heterogenous expression, after that, their biochemical characteristics and the function of catalyzing the biosynthesis of aster groups for DAs lack of aster groups are investigated. In the end, the catalytic sites for key BAHD candidate genes are recognized through bioinformatics analysis and protein fixed-point mutant. Then, the key BAHD genes catalyzing the generation of ester groups for DAs would be identified and the toxicity-formation mechanism of A. carmichaeli would be revealed. Thus, this study will provide the theoretical basis for creation of the safe and efficient germplasm of A. carmichaeli by genetic engineering.

乌头为骨干中药材之一，但剧毒特性严重制约了它的开发和应用。因此，解析乌头毒性形成机理对解决其毒性问题具重要意义。乌头毒性与二萜生物碱骨架上的酯基密切相关，而BAHD酰基转移酶是催化酯基合成的关键终端酶。本项目在种质资源收集的基础上，通过活性成分分析获得主成分富含酯基的剧毒乌头和主成分不含酯基的无毒维西乌头。基于此，拟采用比较转录组手段鉴定二者BAHD家族成员，依据其表达模式和显著富集代谢途径分析，以及BAHD成员系统发育树各分支的功能特征，筛选乌头BAHD候选基因；结合其时空表达特征和主要含酯基和无酯基二萜生物碱的比例变化，锁定乌头关键BAHD候选基因；利用异源表达获得目的蛋白，分析其催化酯基合成功能；通过生物信息学分析和蛋白定点突变揭示关键基因活性催化位点，从而阐明关键BAHD基因催化二萜生物碱酯基合成的机理和乌头毒性形成的分子机制，并为利用遗传改良获得安全高效的乌头新资源提供理论依据。

乌头属是一类剧毒且具有较高药用价值的传统药用植物，二萜生物碱是其主要的毒性和活性物质基础，但生物合成机制尚属空白。为了阐明二萜生物碱的生物合成，通过转录组测序技术对根和叶片组织进行了基因差异表达分析。筛选出二萜生物碱骨架合成途径上的编码酶，如ent-柯巴基焦磷酸合酶（ent-copalyl diphosphate synthases），ent-贝壳杉烷合酶（ent-kaurene synthases），贝壳杉烷氧化酶（kaurene oxidases），环化酶（cyclases）以及关键的转氨酶（aminotransferases）。此外，从附子的转录组测序中发现了负责二萜类生物碱骨架修饰的候选基因，例如单加氧酶 （monooxygenases），甲基转移酶（methyltransferase）以及BAHD酰基转移酶（BAHD acyltransferases）。以主成分富含酯基的剧毒乌头(Aconitum carmichaelii)和黄草乌(Aconitum vilmorimianum)，以及主成分不含酯基的无毒维西乌头(Aconitum weixiense)为材料，以乌头转录组测序注释为BAHD酰基转移酶基因为依据，设计引物分别克隆三种乌头属植物的BAHD酰基转移酶，并进行生物信息学分析，根据BAHD家族成员系统发育树中各分支的功能特征，初步筛选乌头候选BAHD基因；再结合乌头各组织器官不同发育时期BAHD候选基因的时空表达特征，进而锁定与二萜生物碱合成相关的关键BAHD候选基因，并对关键BAHD酰基转移酶基因进行蛋白表达。通过IPTG诱导成功获得各关键候选基因异源蛋白，优化获得最佳表达条件28 ℃、4 h、0.5 mM IPTG；目前酶学催化试验还在进一步进行中。另外，为后期验证基因功能提供植株再生遗传转化体系，本实验以黄草乌种子、腋芽、茎段、叶片为外植体，通过消毒条件的优选、芽诱导、愈伤组织诱导和分化等步骤，构建了黄草乌再生体系。并采用UPLC-Q -TOF-MS对乌头与云南省其他7种乌头属伪品或替代品植物进行了代谢组成分分析。标识化合物之一fuziline对小鼠无急性毒性，提示该生物碱具有较低毒性。本研究获得乌头关键BAHD候选基因，为鉴定其它候选基因及找到乌头剧毒特性关键BAHD基因奠定基础，以期为解决乌头剧毒性及创造乌头低毒高效新种质资源。