薄荷茉莉酸应答转录因子MhMYC2调控精油合成的分子机制研究

Mint (Mentha haplocalyx Briq.) is a traditional Chinese herbal medicine. Essential oil (EO) is the major ingredient of Mint, and EO has multiple pharmacological properties such as anti-oxidative, anti-carcinogenic and so on, so the content of EO is the main indicators to measure the quality of Mint. Therefore, it is of great significance to improve the medicinal quality of Mint by increasing the content of EO. Jasmonate (JA) treatment increases the accumulation of metabolites by inducing the expression of biosynthetic genes involved in secondary metabolism, but the mechanism for JA-mediated EO accumulation remains largely unknown in Mint. The comparative transcriptome analysis and quantitative real-time PCR assays in response to JA treatment showed that JA activated the expression of the biosynthetic genes of EO, and a MYC gene MhMYC2, which was most significantly induced by JA in all MYC genes and was involved in the regulation of secondary metabolism was isolated. MhMYC2 was also co-induced with the biosynthetic genes of EO after JA treatment. These results implied that MhMYC2 regulated EO biosynthesis through responding to JA. In this project, various approaches including subcellular localization, transgenic Mint plants analysis, GC-MS analysis of metabolites, and target genes screening were adopted to verify the function of MhMYC2 and its target genes in Mint and further uncover its regulatory mechanism in JA-mediated EO biosynthesis. This study will make progresses on the transcriptional regulatory mechanism of EO biosynthesis, and provide useful information and gene resource for breeding EO-enriched Mint through genetic engineering. All of these achievements will be significant for boosting the economic value and medicinal quality of Mint.

薄荷是传统中草药，其活性成分精油有抗氧化、抗癌等药效，故精油含量是衡量薄荷药材品质的主要指标，因而提高精油含量对薄荷药材品质改良意义重大。茉莉酸诱导次生代谢产物合成酶基因表达及代谢产物积累，但薄荷中茉莉酸信号调控精油合成的机制未见报道。通过薄荷茉莉酸处理差异转录组分析和荧光定量验证发现茉莉酸激活精油合成酶基因表达；并挖掘到一个受茉莉酸极显著诱导且参与次生代谢调控的MYC基因MhMYC2，它与精油合成酶基因在茉莉酸处理下呈共诱导的模式，推测茉莉酸信号通过激活MhMYC2调节精油合成。本项目拟在薄荷中，通过亚细胞定位、薄荷转基因验证、代谢产物GC-MS分析、靶基因筛选等实验鉴定MhMYC2的功能及靶基因，揭示其参与茉莉酸调控精油合成的机制。本研究结果将在精油合成转录调控机理方面取得突破，为利用基因工程手段培育高精油含量薄荷品种提供基因资源和理论依据，对提升薄荷经济产值意义重大。

薄荷精油有抗氧化、抗癌等药效，也是重要的工业原料。故精油含量是衡量薄荷药材品质和商业价值的关键指标，对薄荷精油合成的调控对提高单位面积内精油含量和提升薄荷药材品质具有重要意义。茉莉酸（JA）应答MYCs基因通过不同的转录调控策略调节次生代谢产物合成酶基因表达，调控代谢产物合成，但薄荷MYCs基因响应JA信号调控精油合成的分子机制至今未见报道。本研究从薄荷JA处理差异转录组数据中筛选到123个bHLH基因，其中4个MYCs基因受JA诱导上调表达。我们克隆了一个受JA诱导最显著的MYCs基因MhMYC2。生物信息学分析发现MhMYC2与SmMYC2、AaMYC2同源，具有保守的JAZ互作结构域、转录激活结构域和核定位信号。基于PCR测序的基因结构分析显示，MhMYC2不具有内含子。基因表达模式分析显示，MhMYC2基因在多数组织中均有表达，茎中表达量最高，并且MhMYC2受JA诱导表达，与精油合成酶基因呈共诱导模式，此外，MhMYC2受ABA、干旱、盐诱导上调表达。亚细胞定位与转录激活实验证明MhMYC2定位于细胞核，具有转录激活活性。此外，我们克隆了7个薄荷MhJAZs基因，蛋白互作研究显示MhJAZs蛋白与MhMYC2或AtMYC2在酵母或原生质体中存在互作。通过基因组步移实验克隆了薄荷精油合成酶基因MhGPPS-SSU和MhGPPS-LSU启动子，其中MhGPPS-LSU启动子包含MhGPPS-LSU-3和MhGPPS-LSU-7两种多态性，元件分析显示MhGPPS-SSU和MhGPPS-LSU-3启动子含G-box或G-box类基序，GUS染色表明MhGPPS-SSU和MhGPPS-LSU启动子具有转录活性。酵母单杂交实验证明MhMYC2和AtMYC2与MhGPPS-LSU-3启动子中CACGTT基序以及MhGPPS-SSU启动子中的CACATG、CACGTG、CATGTG基序互作。Dual-LUC实验证明MhMYC2和AtMYC2激活MhGPPS-LSU-3启动子，表明MhMYC2激活精油合成。同时，我们创制了MhMYC2融合GFP或6xMyc标签的转基因椒样薄荷植株，将进一步结合生理生化研究MhMYC2调控精油合成的调控网络。项目资助发表SCI论文3篇，获省部科技进步奖二等奖1项，正在投稿SCI论文2篇，在申报国家发明专利1项、培养在读硕士研究生1名。