广藿香百秋李醇生物合成的调控相关基因克隆及其功能鉴定

Patchouli alcohol, one of the active constituents of Pogostemon cablin, has been the index component for evaluating patchouli medicinal materials and patchouli oil quality in all of China Pharmacopoeia. Patchouli alcohol has widely been used in pharmaceuticals, food and cosmetics, which gives rise to an increasing demand. It was reported that the content of patchouli alcohol was very little, which limited the large-scale production and development and utilization of P. cablin and patchouli alcohol. Therefore, it is necessary to study the related gene cloning and functional identification of patchouli alcohol biosynthesis in P. cablin. On the basis of the active component’s formation and the dynamic accumulation of patchouli alcohol, this project will establish the transcription group library of ‘Nanxiang’and‘Paixiang’whose patchouli alcohol contents had a big difference by using the transcriptome sequencing, have a screening on the related candidate genes of patchouli alcohol biosynthesis by GO and Pathway biological information analysis. The candidate genes will also be verified. The specific primers will be designed according to the obtained cDNA sequence, and the RACE PCR technology will be used to amplify the full-length sequence from the related gene of the patchouli alcohol biosynthesis. The transgenic systems, positive and interferential expression carriers will be constructed and switched into the patchouli tissue. GC-MS method will be used to determine the patchouli alcohol contents, and the function of target gene in the organism of P. cablin will also be verified by the RNAi expression technology. This project will discuss the molecular regulation mechanism of the patchouli alcohol biosynthesis and provide theoretical foundation and technical support to improve patchouli alcohol content in P. cablin.

百秋李醇是历版《中国药典》规定的用于评价广藿香药材及广藿香油质量的指标成分，广泛应用于药品、食品及日用化妆品行业，需求量与日俱增。由于广藿香中百秋李醇含量低，限制了广藿香、百秋李醇的规模化生产和开发利用，因而有必要对百秋李醇生物合成调控基因展开研究。本项目在前期对广藿香活性成分形成、百秋李醇的动态积累等基础上，利用转录组测序（RNA-seq）技术，构建百秋李醇含量差异大的“南香”和“牌香”叶转录组文库，通过GO、Pathway等生物信息分析，筛选与百秋李醇生物合成调控相关的候选基因，采用实时荧光定量PCR验证；根据已获的cDNA序列设计特异引物，应用RACE PCR技术扩增目的基因全长序列；建立广藿香转基因体系，构建正义及干扰表达载体并导入广藿香植物组织，GC-MS法测定百秋李醇等含量，验证基因功能。本研究将探讨百秋李醇生物合成分子调控机制，为整体提高百秋李醇含量提供理论依据和技术支撑。

百秋李醇作为广藿香的活性成分和广藿香挥发油的主要成分之一，是历版《中国药典》规定的用于评价广藿香药材及广藿香油质量的指标成分。广藿香中百秋李醇的含量低限制其工业化、规模化生产及开发利用。目前关于百秋李醇合成途径中相关酶的分子功能及分子调控机制仍不清楚，要提高广藿香中百秋李醇的产量，就必须了解其在广藿香体内的生物合成调控机制。为此，本课题构建了“南香”和“牌香”茎和叶的转录组文库，筛选与百秋李醇生物合成相关的候选基因，同时分析相关候选基因在不同生长期的表达情况；此外，本课题组克隆FPS、IPI和HMGCR等3个候选基因，对其序列结构、同源性、保守结构域特点进行了系统分析，并验证了IPI和HMGCR基因的功能。“南香”与“牌香”幼嫩茎、叶转录组测序共获得17148个差异表达基因，GO和Pathway功能分析，发现有1913个基因与次生代谢产物合成相关，12个基因与百秋李醇生物合成相关。FPS、PTS、HMGCR基因在成熟期叶片中表达量高于幼苗期，与转录组数据分析结果一致。克隆得到包含1050 bp、879 bp和1278 bp开放阅读框的广藿香FPS、IPI和HMGCR基因片段，分别编码349、292和425个氨基酸。本课题组用HMGCR 68-425bp的C-端序列在大肠杆菌中表达，酶活性GC-MS验证表明广藿香HMGCR具有酶活性，可催化底物合成甲羟戊酸，构建了pTRV2-PcIPI基因沉默和pCAMBIA1300-35S-HMGCR过表达载体，分别导入广藿香植株。IPI基因沉默后会减少百秋李醇的生物合成；而HMGCR过表达，植株中百秋李醇含量显著上升，这表明两个基因均与百秋李醇合成调控相关。此外，我们对广藿香精油的提取分离条件进行了优化，并开发了2种广藿香新产品。本课题探讨了广藿香中百秋李醇生物合成的分子调控机制，为整体提高广藿香中百秋李醇含量提供理论依据和技术支撑。