山鸡椒醇脱氢酶LcADHs基因调控柠檬醛高效合成的分子机制研究

山鸡椒精油是我国传统的天然抗生素来源之一。柠檬醛是山鸡椒精油的主要成分和抑菌的主效成分，其含量是决定精油品质和抑菌产品开发的关键。醇脱氢酶（ADH）是催化柠檬醛生物合成最后一步的关键限速酶，揭示主效ADHs基因调控柠檬醛高效合成的分子机制，对创制稳定高产的山鸡椒高柠檬醛含量新品种具有重要意义。本课题组前期基于山鸡椒基因组和转录组筛选获得在染色体上形成基因簇并在精油合成期高表达的8个关键ADHs基因。在此基础上，本研究拟利用单细胞RNA测序明确山鸡椒果皮合成柠檬醛的关键细胞类型和基因，结合酶活性检测和山鸡椒瞬时转化技术筛选主效ADHs，进一步利用定点突变和基因编辑技术挖掘主效ADHs特异性合成柠檬醛的关键酶活位点，并通过遗传转化主效ADHs创制高柠檬醛含量的山鸡椒新种质。研究结果将揭示ADHs调控山鸡椒柠檬醛生物合成的分子机理，为高柠檬醛含量的山鸡椒新种质选育奠定理论基础和技术手段。

The essential oil of Litsea cubeba is one of the traditional sources of natural antibiotics in China. Citral is the main component of both L. cubeba essential oil and antibacterial substances. The content of citral is the key to determine the quality of the essential oil and the development of antibacterial products. Alcohol dehydrogenase (ADH) is the crucial rate-limiting enzyme that catalyzes the last step of citral biosynthesis, revealing the molecular mechanism of the major ADHs gene regulating the efficient synthesis of citral, which is of great significance for increasing the content of citral and creating stable and high-yielding new germplasm. We identified 8 key ADHs genes according to the L. cubeba genome and transcriptome. On this basis, the study intends to use single-cell RNA sequencing to clarify the key cell types and genes for citral biosynthesis from the peel of L. cubeba. Then the enzyme assays and transient expression of key ADHs genes will be carried out to identify the major ADHs. Furthermore, we will excavate the key enzyme sites of major ADHs that determine the specific synthesis of citral via site-directed mutagenesis and CRISPR/CAS9. A citral-synthesis regulatory network with ADHs as the core will be constructed, and a new germplasm of L. cubeba with high citral content will be created through genetic transformation of ADHs. The results of the study will clarify the molecular mechanism of ADHs regulating the citral biosynthesis in L. cubeba, which will provide materials and molecular assisted selection method for citral content in L. cubeba breeding.