黑暗和机械刺激影响花生荚果膨大发育的分子机理研究

A characteristic feature of peanut is ‘aerial flower and subterranean fruit’, in which the gynophore drives the developing pod into the soil for subsequent development underground. Failure to penetrate into soil inhibits ovary enlargement, leading to yield loss of peanut. Previous studies showed that darkness and mechanical stimulus are essential external conditions for the formation of pod. However, the molecular mechanism of subterranean pod formation is far from being understood. We have identified eight genotypes of peanut whose pods, even not penetrate into soil, may start swelling aerially. In this study, we are going to re-sequence these genotypes of peanut and identify SNPs potentially related to pod swelling via comparison of genomes from the eight genotypes. Besides genomic analysis, we will also be employing transcriptomic and proteomic approaches to understand the mechanism of pod swelling under darkness and/or mechanical stimulus conditions. The effect of dark and mechanical stimulus will be studied by covering aerial gynophore with black plastic sleeves and transparent pearlite particles as follows: (1) transparent sleeves as control; (2) black sleeves or aluminium foil sleeves for darkness without a mechanical stimulus; (3) transparent sleeves with transparent pearlite particles for mechanical stimulus under light; (4) black plastic sleeves with transparent particles for darkness plus a mechanical stimulus. Samples will be collected 0, 5, 10, 20 days after treatment. Endogenous hormone contents will be measured, and total RNA and proteins will be isolated using corresponding methods. RNA-sequencing will be performed using HiSeq2500 platform, and proteomics analysis will be conducted using iTRAQ. These multidimensional omics data will be used for construct a comprehensive database, which, in turn, will be used for multi-omics analysis in order to advance understanding of molecular mechanism of pod enlargement. The results obtained in this study will pay a way for understanding of pod enlargement under darkness and/or a mechanical stimulus, and also facilitate the development of high-yield cultivars in peanut.

花生是植物界少有的“地上开花，地下结果”的植物，受精后雌蕊柄向地伸长将子房带入地下完成荚果发育，未入土的果针不能膨大形成荚果，从而影响花生产量。研究表明黑暗和机械刺激是影响荚果膨大的两个基本因素，但其分子机理至今未明。课题组前期开展了针对黑暗和机械刺激影响荚果膨大的预实验，并从近3000份资源中筛选出8份与荚果膨大相关的材料。基于此本项目拟通过对不同基因型材料重测序，挖掘与荚果膨大相关的SNP位点。通过转录组、小RNA和蛋白组以及内源激素测定，分析在果针不同处理（正常、黑暗、机械刺激和黑暗+机械刺激）条件下荚果膨大相关的差异表达基因、蛋白和miRNA以及激素变化等。最后采用多组学联合分析建立转录、转录后调控、翻译水平的调控网络，从多维度揭示黑暗和机械刺激影响花生荚果膨大的分子机理。本研究对解析花生独特果实发育模式具有重要的理论意义，并将丰富花生荚果遗传改良的分子理论，具有潜在的育种前景。

本项目通过基因组、转录组、miRNA和蛋白组以及内源激素测定，分析了荚果膨大相关的差异表达基因、蛋白和miRNAs，以及果针在不同处理条件下（正常、黑暗、机械刺激和黑暗+机械刺激）的激素变化，从多维度揭示了花生荚果膨大的分子机理。首先通过二代和三代高通量测序相结合对地方品种“伏花生”进行了全基因组de novo测序，获得长度为2.55 Gb的花生栽培种基因组，覆盖预测的花生基因组的~97%。花生栽培种全基因组测序的完成为后续的多组学联合分析提供重要的参考基因组，是后续分析顺利开展的重要前提。在不同处理条件下（正常、黑暗、机械刺激和黑暗+机械刺激）的荚果膨大结果发现花生荚果在模拟黑暗和机械刺激条件下发生膨大，其中机械刺激对花生荚果膨大的作用大于黑暗条件的作用。不同处理条件及花生荚果膨大的不同阶段中果壳的植物激素含量均存在差异，表明植物激素可能在花生荚果发育过程中起着重要的调控作用。通过对花生荚果不同发育时期的果壳和种子进行 small RNA 测序，共获得62个新的花生miRNA和203个已知的miRNA，发现miRNA及其调控的目标基因在荚果发育过程中呈动态变化，表明不同的 miRNA 在花生荚果、种子发育的不同时期发挥作用，该结果为阐释花生荚果暗发育的分子机制提供了转录后调控的理论参考依据。基于已发表的花生组学结果，构建了花生基因蛋白质组学联合分析平台和花生脂质组学分析平台。此外，花生基因组测序结果中发现，与其他作物基因组相比，FAR1基因家族在花生基因组中富集，且在物种进化过程中经历了更强烈的自然选择过程。这些研究结果为深入解析花生独特的果实发育现象提供线索，对研究植物果实器官的起源和进化具有重要的理论意义，并将丰富花生荚果遗传改良的分子理论，具有潜在的育种前景。本项目发表论文8篇，其中SCI收录论文7篇。项目组2名成员职称获得晋升。