miR168b介导Δ9D基因调控沙棘果肉高积累棕榈油酸的机理研究

Sea buckthorn (Hippophae L.) is an important economic tree for desertification prevention and soil and water conservation, and there is an increasing demand globally for sea buckthorn pulp oil rich in palmitoleic acid (omega-7, 25-48%). The delta 9 desaturase (Δ9D) gene, which catalyzes the conversion of C16:0 to C16:1, is the critical gene involved in palmitoleic acid biosynthesis, but the post-transcriptional regulatory mechanism of miRNA in sea buckthorn pulp oil containing high content of palmitoleic acid has not been elucidated. We detected the Δ9D gene was targeted by miR168b in our previous work. Based on our already available sea buckthorn germplasms with high diversity in palmitoleic acid content, and the sea buckthorn pulp of different developmental stages will be selected as experiment materials. We will use GC-MS, qRT-PCR and ddPCR to reveal regulatory relationship between miR168b and Δ9D gene, and the regulatory role of ‘miR168b-Δ9D’ module in palmitoleic acid biosynthesis and accumulation according to the palmitoleic acid content in pulp and the expression of miR168b and Δ9D. By over-expression and silence approach, it will be reveal led the molecular mechanism of miR168b targeting Δ9D gene regulates high accumulation of palmitoleic acid in sea buckthorn pulp based on the comparative analysis of palmitoleic acid contents and miR168b and Δ9D gene expressions between transformation organization and control group. This project will provide first revelation the regulatory mechanism of miR168b targeting Δ9D gene regulates high accumulation of palmitoleic acid in non-seed tissues of plant. This will provide great significance to improve the palmitoleic acid composition in sea buckthorn.

沙棘是荒漠防治和水土保持的重要经济树种，富含棕榈油酸(omega-7脂肪酸)(25-48%)的果肉油的全球需求量正日益增加。Δ9D基因与棕榈油酸合成密切相关，但其miRNA调控机制仍不清楚。前期研究发现沙棘miR168b靶向Δ9D基因，本项目以近缘、棕榈油酸含量存在明显差异的多对沙棘种质不同发育期果肉为材料，采用GC-MS、qRT-PCR和ddPCR等方法，通过果肉棕榈油酸含量及miR168b、Δ9D基因表达的分析，明确miR168b与Δ9D基因间的调控关系及其在棕榈油酸合成积累过程中的作用；利用过表达和沉默miR168b与Δ9D基因的方法，比较转化组织中棕榈油酸含量及miR168b、Δ9D基因表达与对照的差异，揭示miR168b介导Δ9D基因调控果肉高积累棕榈油酸的作用机制。这将首次揭示“miR168-Δ9D”模块调控植物非种子组织高积累棕榈油酸的机理，对高棕榈油酸沙棘育种具有重要意义。

沙棘是具有重要营养价值和生态价值的木本油料作物，因其果肉(非种子组织)中富含棕榈油酸(omega-7脂肪酸，C16:1n7) (25-48%)而在世界范围内得到广泛应用。Δ9D基因与棕榈油酸合成密切相关，但其miRNA调控机制仍不清楚。前期研究发现沙棘miR168b靶向Δ9D基因，本项目以近缘、棕榈油酸含量存在明显差异的不同沙棘种质(‘杂56’和‘TF2-36’)不同发育期果肉为材料，采用GC-TOF/MS、qRT-PCR和非靶向代谢组学分析表明，沙棘果肉发育期间Δ9D基因表达量与棕榈油酸含量呈正相关，而且16:1-CoA、G3P的高丰度与Δ9D、GPD1基因的高表达协同调控果肉高积累棕榈油酸，表明沙棘Δ9D基因在棕榈油酸合成过程中具有重要作用。通过烟草遗传转化验证发现，过表达miR168b的靶基因Δ9D可显著提高烟草叶片棕榈油酸含量；过表达miR168b的烟草叶片Δ9D基因表达量和棕榈油酸含量均显著降低；在稳定转化Δ9D基因的烟草中，分别沉默和过表达miR168b对叶片棕榈油酸合成具有明显的促进和抑制作用，表明miR168b负调控靶基因Δ9D而影响棕榈油酸的合成。本项目明确了miR168b-Δ9D模块在沙棘果肉棕榈油酸合成过程中的作用，研究结果不仅为深入解析非种子组织油脂和重要脂肪酸合成代谢机制提供了新的科学依据，而且对高油高棕榈油酸沙棘育种具有重要意义。