印楝素生物合成途径关键CYP450基因筛选与功能分析

Due to the high efficient and broad-spectrum insecticidal activity, azadirachtin, a class of active plant ingredient which is major derived from the seeds of Azadirachta indica, is regarded as an valuable non-timber forest product and can develop ideal botanical pesticides. To date, it is not mature to manufacture azadirachtin in large scale using genetic engineering methods, as a consequence of scientific studies related to the downstream pathway and key enzyme genes in azadirachtin biosynthesis are still insufficient. This project adopts phytochemistry, bioinformatics and molecular biology technologies to study the azadirachtin biosynthesis pathway, and major concern is the oxidation process of azadirachtin backbone. By the means of full-length isoform sequencing combined transcriptomic analysis on different tissues, coupled with the azadirachtin content data, key cytochrome P450s involved in azadirachtin biosynthesis will be screened. Subsequently, overexpressing and RNAi technologies will be accomplished in neem hairy root platform to deduce the function of CYP450s. The results will be able to demonstrate the process of azadirachtin oxidative modification and key enzyme genes for the first time, and can further contribute to the regulation of azadirachtin metabolic network. This project will have important scientific significance in constructing the azadirachtin bioreactor, breeding a new species of high azadirachtin content neem, improving the utilization value of forest resources and implementing the forest sustainable management.

印楝素是一种主要来源于林木印楝果实的植物活性成分，具有广谱高效的杀虫效果，是极具经济潜力的非木质林产品，能开发出理想的植物源农药，市场需求巨大。但是目前关于印楝素生物合成下游途径的研究基本处于空白，涉及的关键基因也不明确，严重阻碍了印楝素生物合成及代谢调控研究的深入。申请项目拟通过植物化学、生物信息学和分子生物学技术研究印楝素的生物合成途径，重点探讨印楝素骨架的氧化加工过程，通过全长转录组测序和不同组织转录组差异分析，结合印楝素在的组织间含量差异，筛选正相关的CYP450s，并利用印楝毛状根平台采用基因过表达和RNAi技术，结合HPLC-Q-TOF-MS检测鉴定其功能。该研究结果能明确印楝素生物合成途径中的氧化修饰过程及关键酶基因，对于印楝素生物合成途径改造和代谢网络调控、构建印楝素生物反应器、开发高印楝素含量的印楝新品种、提高森林资源利用价值、进行森林可持续经营具有重要科学意义。

印楝是一种原产于印度、马来西亚和印度尼西亚等国的常绿乔木，具有药食两用的特性。研究人员发现印楝种仁中含有一类四环三萜类化合物——印楝素，活性评价发现其具有显著的驱虫效果，能有效防治小菜蛾、菜青虫和蝗虫等多种农业害虫，是一种理想的植物源杀虫剂。此外，用印楝叶片开发出的保健品具有消食、瘦身和治疗糖尿病等作用，已经在市场上被广泛使用。然而受限于植物资源，印楝素及其相关产品产能明显不足，市场缺口巨大。因此研究印楝素的生物合成途径，构建高产印楝素生物反应器是解决这一问题的关键。本研究通过全长转录组和二代转录组数据对不同组织样品进行分析，寻找差异转录本，结合基因组数据及组织间化学成分差异筛选出6条印楝素合成相关CYP450基因。