油棕miRNA5179介导的表达调控对果实发育的影响

Oil palm(Elaeis guineensis Jacg) was the most productive oil-yield crop in plant kingdom, accumulating up to 90% oil in its mesocarp. Palm oil accounts for 33% of vegetable oil and 45% of edible oil in the world. Fatty acids (FAs), which were accumulated in the form of triacylglycerol as major storage components, have diverse uses contributed to palm oil many food and industrial traits.Knowledge of fruit development was highly considered as an essential manner to understand the machanism of FAs and oil synthesis in fruit. However, study of regulatory mechanisms of fruit developmet in palm oil was rare, even for microRNA(miRNA), which was approximately 18 to 25 nucleotides long and produced from hairpin-shaped precursors, regulating genes expression in eukaryotes by targeting mRNAs for translational repression or cleavage.In our previous study, a comprehensive picture of the expression of mRNA and miRNAs in oil palm mesocarp were presented and highlights AGL2 subfamily was the most abundant during mesocarp development.With bioinformatics analysis, eg-miR5179 was predicted to target one MADS-box gene,which belongs to EgAGL2-1(AGL2 subfamily).Further confirmation of the relationship between eg-miR5179 and MADS genes showed their expression levels were exhibited negative correlation during maturation of oil palm fruit.Since MADS members were recently reported to play a key regulatory role on fruit ripening. It was implied these MADS transcripts might be involved in the fruit development and oil synthesis of oil palm nut. To evaluate the effect of eg-miR5179 mediated MADS gene on oil palm nut development, we will try to characterize the function of MADS gene and regulatory mechanism of miR5179 via a variety of mutant model plant lines，such as Arabidopsis thaliana, in which overexpression of eg-miR5179, MADS (target gene) and co-expression of two of them,and so on.QPCR, microscope analysis, measurement of seed weight and components analysis were performed and compared between WT and mutant lines. Knowledge of this new pathway that eg-miR5179 modulating MADS-box gene was involved in fruit development would provid insight into the important mechanisms of oil palm mesocarp maturation and facilitate to screen molecular markers in the selection of new varieties with higher yields.

油棕为典型的多年生油料植物，是海南最具特色的热带经济作物。研究者在前期油棕果实发育转录组和microRNA表达谱的分析中发现，油棕中果皮中AGL2家族是整个果实发育过程中表达最为丰富的一个家族，其表达水平与油棕果实的发育和成熟相一致。同时，在整个油棕果实发育过程中，eg-miR5179的动态变化趋势正好与其预测靶基因MADS-box呈明显的负相关。而预测得到的靶基因MADS-box正是AGL2家族的成员之一。为了寻求并阐明油棕果实发育相关的MADS-box及其上游重要调控因子参与油棕果实发育的分子机制，本项目拟通过针对油棕中已克隆得到的eg-miR5179与MADS-box基因克隆、构建植物表达载体和遗传转化相结合的方法对此代谢通路进行鉴定和分析，在此结果上阐明由eg-miR5179介导的调控通路在油棕果实发育过程中作用机制，并为最终探究该通路在油棕遗传改良中的潜在价值提供研究基础。

油棕（Elaeis guineensis Jacg），别名油椰子，属棕榈科油棕，为典型的热带油料植物，因其果实含油量高以及单位面积产油量大而被誉称为“世界油王”。油棕已成为国际上第二重要油脂原料。以往对油棕油脂代谢的分子机制已开展了大量研究，但油脂生物合成的调控机制仍未充分解析，特别是关于microRNA的调控作用。前期研究中发现油棕的一条保守性microRNA-miR5179（EgmiR5179），在油棕发育过程中的动态表达与油棕油脂积累趋势相一致。构建异源过表达EgmiR5179拟南芥植株（OXmiR5179），检测发现其种子中油脂含量提高，特别是亚麻酸（18:3）积累增加；而碳水化合物和类黄酮含量减少。生物信息学预测EgmiR5179的靶基因为NAD+ Transporter 1（NDT1）。5’ RACE，双荧光报告和实时荧光定量（Quantitative Real-time PCR）实验共同证实了EgmiR5179在油棕和拟南芥中的靶基因均是NDT1。NDT1是NAD+ 的运输者，负责运输NAD+从胞浆进入线粒体内膜。研究发现，OXmiR5179拟南芥转基因株中NAD+含量以及NAD/NADH的比值均呈现下调。由此推测，EgmiR5179通过抑制NDT1蛋白表达，从而减低NAD+转运进入线粒体，阻断了NAD+依赖的β氧化，延迟脂肪酸从油脂中释放，最终导致油脂积累增加，特别是不饱和脂肪酸亚麻酸(C18:3)升高。同时，线粒体中NAD+减少，光合作用减弱，引起淀粉和类黄酮生物合成缓慢。本研究首次报道了EgmiR5179在植物细胞内代谢产物的生物合成中发挥着一条重要的调控通路：EgmiR5179通过抑制NDT1，下调NAD+含量和NAD/NADH比值来参与油脂、脂肪酸、淀粉和类黄酮生物合成的调控。由此解析了EgmiR5179在油棕中果皮发育后期升高的内在原因。这条通路提供了从非编码RNAs调控的角度认识油脂和次生代谢产物生物合成代谢的分子机制的新思路。EgmiR5179有望今后成为基因代谢工程的分子靶标，用于遗传改良油棕中果皮以获得富含油脂和特定脂肪酸的高价值油品。