油棕脂肪酸合成代谢相关miRNAs的发掘及其调控机制研究

Oil palm (Elaeis guineensis Jacg）is a perennial tropical tree crop and the highest oil producing plant. This fruit is usually in producing two valuable vegetable oils: palm oil and palm kernel oil. Among them, palm oil, extractable from the outer flesh (mesocarp) of oil palm, is second only to soyabean oil in the total world's production of vegetable oils. MiRNAs, which are approximately 18 to 25 nucleotides long and produced from hairpin-shaped precursors, regulate genes expression in eukaryotes by targeting mRNAs for translational repression or cleavage. It has been demonstrated that miRNAs exist extensively in plants with high conservation and have crucial regulatory functions in diverse biological processes, including plant development and seed formation, yet their population and function of fatty acid anabolism in fruit of oil palm are still not well understood, especially at different developmental stages and tissues with varied oil contents. In our previous studies, we systematically sequence and bioinformatics analyze the transcriptome of oil palm mesocarp and found many genes are significantly different expression among five development phases. These results strongly imply that, to some extent, there maybe exist some regulatory factors acting on these genes which invovled in fatty acid biosynthesis pathways during the course of the mesocarp development. On this basis, we next to illustrate two issues: 1) miRNA expressed specifically in mesocarp: Solexa sequencing and bioinformatics analysis were used to excavate the small RNA for the mesocarps of oil palm fruits with five developmental stages, and miRNA expression profiles, especially novel miRNAs, specific to the mesocarp with the specifical development phase, can be discovered; 2) miRNAs involved in fatty acids anabolism: base on the genomewide analysis of miRNA profiles, the most different significantly expressed miRNAs, especially which involved in the fatty acids biosynthesis, were screened, after prediction of their target genes. Validation of negative relationship of expression levels between specific miRNAs and their target genes in the oil palm mesocarps by qRT-PCR and Northern blot,and identification their regulatory roles through detection the composition and proportion of fatty acids in transgene model plants, such as Arabidopsis thaliana, which transfected with miRNAs, target genes and dot-mutation target genes, respectively, by Gas Chromatography technology. Base on these results, the spatio-temporal pattern of the miRNAs expression and the regulation mechanism with the special miRNAs, including known and novel miRNA cascade, in oil palm fatty acid anabolism pathways can be clarified. Knowledge of the miRNA regulatory mechanism in oil palm fatty acid anabolism is quite essential to modify the composition and proportion of oil palm fatty acids for improving their nutritional value and function properties in various oil applications.

油棕（Elaeis guineensis Jacg），为典型的热带油料植物，是海南最具特色的热带经济作物之一。业已证实，MicroRNAs(miRNAs)在植物许多重要性状相关的功能基因调控网络中发挥重要作用，但在油棕脂肪酸合成代谢中的调控作用还不清楚。在前期研究中，我们以油棕果实为材料，开展了转录组测序工作，获得了不同发育时期差异表达的功能基因。在此基础上，拟开展两方面的研究:1）开展大规模油棕的中果皮miRNA的发掘，获得其中新的、特异表达的miRNA；2）在大规模测序的基础上筛选脂肪酸代谢相关miRNA，预测其靶基因，通过遗传转化方法对miRNA与相关靶基因进行功能验证，探究miRNA表达对脂肪酸生物合成的影响, 揭示相关miRNA 在植物脂肪酸代谢途径中的调控机理。本研究的进行将有利于进一步理解油棕脂肪酸代谢的分子调控机理，为今后针对性地改良脂肪酸合成提供重要的物质基础和技术平台。

油棕是典型的热带油料植物，是海南最具特色的热带经济作物之一。针对MicroRNAs(miRNAs)在油棕果实脂肪酸代谢和发育过程中的调控研究仍是空白,本研究以5个不同发育时期油棕果实中果皮为材料，开展了以下工作：（1）转录组和数字表达谱测序工作，获得了不同发育时期差异表达的功能基因；（2）小RNA测序工作，筛选出与中果皮发育相关的miRNA及表达谱，重要的是获得一批新的，果实发育特异性的miRNA；（3）降解组测序工作。miRNA通过作用靶标基因发挥调控作用，由此，本研究结合生物信息学和降解组数据，针对差异表达的miRNA进行靶基因预测分析，挖掘出一系列与脂肪酸代谢和果实发育相关的miRNA。通过实时荧光定量PCR、Northern blot 和5’RACE等方法分别对新miRNA的真实性，miRNA和靶基因的表达模式及切割关系进行验证。整合miRNA和mRNA表达谱，展现出miRNA在油棕脂肪酸代谢和果实发育途径中的调控网络关系。项目研究创新性地建立了油棕果实发育全生育期的miRNA和功能基因表达谱数据库，为今后进一步克隆影响脂肪酸代谢和果实发育的关键miRNA提供依据，从分子水平上改良脂肪酸特性和果实发育提供更可靠的靶标；也为更全面地揭示油棕脂肪酸代谢和果实发育的调控机制提供新视角。