山杏种子油脂累积及脂肪酸组分转化的分子调控机制

Siberian apricot (Prunus sibirica L.) expected to a new type of woody biomass resources is an excellent native species. With the pre-screening, two Siberian Apricot Germplasms with the significant differences of the oil content and fatty acid composition as experimental materials are used to comparatively analysis for seed oil content, fatty acid composition and metabolomics in different developmental stages, and then the pattern of the accumulated oil and the transformed fatty acid composition and its relationship with the metabolite changes are explored. Uniformity digital gene expression (DGE) library is established and Illumina-Solexa deep sequencing is conducted subsequently, and isobaric tagging for relative and absolute quantitation (iTRAQ) is used to identify the dynamic profiles of proteins for developing seeds of Siberian apricot, which contribute to determine the Siberian apricot functional genes and metabolic pathways of the accumulated oil and the transformed fatty acid composition. Simultaneously, the metabolites and the transcripts of transcription factors and metabolic enzymes involved in oil accumulation and the conversion of fatty acid composition are analyzed in different developmental stages. Also the cloning of key oil accumulation-related genes, genetic transformation for Arabidopsis thaliana and expression analysis are performed. Our findings would help to elucidate molecular regulatory mechanism of increased oil production in developing Siberian apricot and to lay an important foundation for some economic oil crops and forest biomass improvement through molecular breeding in the future.

山杏是我国优良乡土树种,有望成为新型木本生物质资源。本项目以前期筛选的2份油脂及脂肪酸组分差异明显的山杏种质为试材，开展其不同发育期种子油脂含量及脂肪酸组成测定与代谢组学的比较分析，从中探究油脂累积及脂肪酸组分转化模式及其与代谢物变化间关系；构建均一DGE文库,开展Illumina-Solexa深度测序分析, 并运用iTRAQ蛋白质组学技术对供试山杏样品进行定量分析，进而确定山杏种子高品质油脂累积途径中关键调控基因及代谢路径；同时,开展高油脂密切相关代谢物的测定分析，并对不同发育期种子中调控脂肪酸累积相关的转录因子或代谢酶基因的转录表达进行检测与分析, 选择开展具有代表性关键调控基因的克隆及遗传转化拟南芥和表达检测分析研究，进而从转录组学、蛋白组学、代谢组学及代谢通路等不同层次阐明山杏种子高品质油脂累积的分子调控机制,这可为分子改良某些有经济价值的油料作物及生物质林木奠定重要基础。

山杏是我国丰富优良乡土树种,有望成为新型木本生物质资源，而其种子油脂累积分子调控网络机制至今尚未明确。本项目以前期证实的2份油脂及脂肪酸组分差异明显的山杏种质（AS-80、AS-30）为试材，开展不同发育杏仁的重量、产量及其油脂与脂肪酸组分含量的测定分析，从中探究山杏种子生长发育与油脂累积及其组分动态变化规律；开展基于脂肪酸不饱和度的生物柴油特性预测模型研建与生物柴油制备及各种燃烧性能评价和种子最佳采摘时期分析确定，结果表明山杏种质AS-80是理想生物柴油原料，其种子最佳采摘期为60 DAF。同时，以供试2份山杏种质不同发育种子为试材，开展DEG文库构建与mRNA转录组测序分析，并以优良种质AS-80的代表性发育期杏仁为试材，开展iTRAQ蛋白质组定量分析与miRNA转录组测序分析；通过对山杏发育种子油脂累积中差异蛋白质与差异表达基因间的关联分析、差异表达miRNAs及其靶基因的筛选确定，结合油脂累积模式研究结果，从多组学层面上有效确定与山杏优质种源AS-80种子高油脂累积密切相关的功能基因（SAD6、FATA、GPAT9、LPAT2、DGAT1等）或调控因子（如ABI3、WRI1和LEC2等）和42个miRNAs及其靶基因，构建出miRNA介导的山杏杏仁发育与油脂累积的调控网络模式、以及转运蛋白介导的碳源分配及能量来源的高油脂合成累积的代谢网络调控模式，进而从转录组学、蛋白组学及代谢网络通路分析等不同层次阐明山杏种子高品质油脂累积的分子调控机制。另外，开展基于mRNA和miRNA测序的油脂累积相关的SNP位点分析，从5个功能基因（ABI3、SAD6 、DGAT1，FAD2和LEC2）和3个pre-miRNA（pre-miR395、pre-miR164b和pre-miR172b）中确定出SNP位点；选择具有代表关键调控基因 (如转录因子WRI1和Ps-pri-miR156a基因)，开展相关基因的克隆及遗传转化拟南芥和表达分析（油脂含量、脂肪酸组分变化、功能基因表达qRT-PCR检测），探究其调控山杏种子高品质油脂累积的作用机制。上述研究结果可为山杏优良种质资源挖掘利用提供科学依据，并可为分子改良某些有经济价值的油料作物及林木生物质资源奠定重要基础。