沙棘果不同器官油脂合成、积累与分配的分子机制研究

The increasing demand for bioactive oils, which are being widely used in food, feed, health-promoting and medical products, can only be met by increased oil production. One way to achieve this is to increase oil yield of oil crops through manipulation of metabolic factors and processes that regulate oil biosynthesis and accumulation in oil-bearing non-seed tissues and seeds. Despite a detailed understanding of metabolic pathways in the oil-accumulating tissues (usually seeds), our knowledge on metabolic control of oil accumulation is limited, and there is virtually no information on regulatory interaction of oil production between non-seed tissues and seeds...Sea buckthorn (Hippophae L.) is a widely distributed woody shrub or small tree with an unusual ability to thrive in unfertilized habitats and the arid and semi-arid areas that are presently increasing due to global warming. This species contains bioactive oils both in the seeds (omega-3 and omega-6 acids) and in the berry pulp (high levels of omega-7 acid). These oils are bioactive per se and also rich in other bioactive compounds with powerful nutritional and medicinal properties, and are being used worldwide to an increasing extent. Presently, the demand for sea buckthorn oils in the world exceeds 3000 tons per year, but output is only about 1800 tons per year. One way to increase sea buckthorn oils availability is to unravel biological mechanisms of oil biosynthesis and accumulation in pulp and seeds, which presently amounts to only 7–11% in the seeds and 1–5% in the pulp...The main objective of this project is to create an advanced innovative multi-technique platform, which can improve our understanding of oil biosynthesis and accumulation and enable identification of key genes for future use in cultivar improvement. Based on our already available sea buckthorn germplasm with high diversity in oil contents and components, including some accessions with unique seedless berries, we will use untargeted (GC-MS, LC-MS and NMR) and targeted metabolomics strategies and integrate these with qRT-PCR analyses and enzyme assays to i) identify key metabolic, biochemical and enzymatic processes and factors with a major role in oil biosynthesis and accumulation in pulp and seeds, especially regulatory systems that control allocation of oil contents and components (especially omega-3, omega-6 and omega-7) between these two tissues, and ii) to identify the key genes that regulate high-content accumulation of omega-7 acid in pulp...This will produce new knowledge about the metabolic factors and processes that are responsible for oil biosynthesis and accumulation in non-seed tissues and seeds for insufficiently investigated woody oil sea buckthorns, especially the regulatory system that controls carbon partitioning between these tissues, and also provide the potential for selecting and breeding superior cultivars of oil sea buckthorns with high-oil non-seed tissues (pulp) and seeds.

沙棘是防风固沙和水土保持的重要经济树种，富含生物活性成分的沙棘油(种子和果肉油)的全球需求量正日益增加。为提高种子(7-11%)和果肉含油量(1-5%)，需系统而深入地研究其合成、积累和分配的调控机制。本项目以亲缘关系近、油含量差异明显的多对种质及无籽沙棘的不同生长发育期果实为材料，采用非目标(GC-MS、LC-MS和NMR)和目标的代谢组学、油合成相关基因表达的qRT-PCR及酶活性分析相结合的方法，鉴定种子和果肉油合成与积累的关键代谢物、代谢途径和调控位点，解析特异表达基因和酶在种子和果肉油形成中的作用，揭示油含量及其重要脂肪酸组分在种子和果肉间分配的调控机制，鉴定克隆调控果肉高积累omega-7脂肪酸的关键基因。这将首次阐明油含量及其重要脂肪酸组分在非种子组织与种子间分配的分子调控机制，可为理解木本油料植物的油合成和积累提供新依据，对种子和果肉油含量均高的双高型沙棘育种具有重要意义。

沙棘是防风固沙和水土保持的重要经济树种，富含生物活性成分的沙棘油(种子和果肉油)的全球需求量正日益增加。为提高种子(7-11%)和鲜果肉含油率(1-5%)，需系统而深入地研究其合成、积累和分配的调控机制。项目以亲缘关系近、种子和果肉含油率差异明显的多对种质为材料，开展了目标和非目标代谢组学、转录组学和比较蛋白质组学研究。RNA-seq和qRT-PCR分析表明，沙棘非种子组织(果肉)和种子油脂合成积累的显著差异源于源基因GPD1和汇基因DGAT1协同表达的差异，调控沙棘果肉和种子富集不同脂肪酸的关键基因是KASII基因，种子中KASII基因高表达为亚油酸和亚麻酸的富集提供了充足底物C18:0，果肉中KASII基因低表达协同FATB和Δ9D基因高表达促进了棕榈酸和棕榈油酸的富集；获得了9个调控沙棘种子和果肉油合成与积累的关键革靶基因元件; miRNA-seq、RNA-seq和qRT-PCR分析，获得137个已知和264个新沙棘miRNA，其中19个（4个已知和15个新miRNA）涉及油脂合成，包括miR168b靶向Δ9D、 novelmiRNA23靶向GPD1、novelmiRNA108靶向ACC、novelmiRNA58靶向DGAT1、novelmiRNA191靶向DGAT2；构建了沙棘种子发育的 miRNAs-靶标转录因子调控网络，发现了多个具有调控作用的功能模块，其中12个在种子发育和油脂合成积累过程中起协同调控作用。比较转录组学研究结果表明，果实发育前期PDAT的作用更为明显，这有利于提高果实甘油三酯中不饱和脂肪酸的比例，发育后期DGAT显著上升，有利于完成脂肪酸和中间产物向甘油三酯转变；基于LC-MS/MS的沙棘果肉发育期间的非靶向代谢组学分析表明，磷脂酰胆碱是影响沙棘果肉油脂合成积累的重要差异代谢物。本项目建立了沙棘种子和非种子油脂合成积累及重要脂肪酸形成分子机制解析的整合组学技术，研究结果不仅阐明了油脂及其重要脂肪酸组分在非种子组织与种子中形成积累的分子调控机制，而且对种子和果肉油含量均高的双高型沙棘育种具有重要意义。发表研究论文27篇，其中SCI收录7篇(1区1篇、2区2篇）、EI收录2篇、CSCD中文核心17篇；获授权沙棘新品种4个(晚黄、晚霞、朝阳和高油1号)；培养博士1名，硕士4名。