基于蛋白质组差异分析的油棕种子萌发机理研究

Oil palm is a tropical oil plant with the highest production efficiency in the world, and is one of the key support objects in the Twelfth Five-Year Plan for Industrial Development of Tropical Crops in China. Oil palm propagates by seeds, but commercial seeds are hybrids, which are complicated to produce and cost much, moreover, with long germination period (60-90 days) and low germination rates (50%-70%), while the germination mechanism remains unclear and current regulating effects stay unsatisfactory. When engaging in sprouting techniques, we found that there maybe one or more critical points during the germination process. Based on the observation, the present work intends to find out these critical points first by investigating the dynamic changes in the cell structure and typical inclusions, for instance, storage protein, then to screen out 2-3 candidate genes that related to these important germination phases by comparing the proteomic changes among these critical points through two-dimensional gel electrophoresis. Furthermore, changes in their mRNA levels during seed germination will be determined by real-time PCR; meanwhile, their biological functions will be verified in Arabidopsis thaliana. Finally, the mechanism of oil palm seed germination will be summarized, to provide theoretical basis to the studies on dormancy breaking and regulating techniques of oil palm seed germination.

油棕是世界上生产效率最高的产油植物，是我国热作产业发展"十二五"规划的重点支持对象之一。油棕通过种子繁殖，商业化种子杂交制种程序繁琐、生产成本较高，萌发周期长（60-90天）且萌芽率低（50%-70%），其萌发机理尚不明确、萌芽调控效果很不理想。我们在从事油棕种子催芽技术研究时发现，该萌发过程可能存在一个或多个临界点。基于此，本项目拟首先通过考察种子组织细胞结构和贮藏蛋白等标志性内含物的动态变化，初步定位油棕种子的萌发临界点；然后以此为突破点，通过双向电泳比较临界点前后及未萌发种子蛋白质组的变化，并通过生物信息学的分析与比较筛选出2-3个在油棕种子萌发的重要阶段起关键调控作用的基因；进一步通过实时定量PCR验证其mRNA表达量在种子萌发过程中的动态变化，同时以模式植物拟南芥为平台对候选基因的生物功能进行验证，从而探索油棕的种子萌发机理，同时为打破种子休眠和促进萌发调控技术研究提供理论依据。

油棕种子是典型的顽拗型种子，种子萌发周期长且萌芽率低，调控机理复杂。本项目同时以种子热处理催芽（含胚乳）胚样品和无菌组培（无胚乳）胚样品为实验材料，通过检测萌芽前后的蔗糖含量变化发现，内源蔗糖可能是胚萌发前期的主要能量来源，而后期必须依靠外源能量维持继续生长发育；基因表达谱分析结果显示，萌发后的胚样品（SP1、SP2、SP3）与对照样品（T0）相比较，上调表达基因均超过5500个、下调表达基因均超过3000个，而这3个样品间的差异基因很少，表明种子催芽阶段涉及到的基因数量很大、生物过程复杂；蛋白质组鉴定分析结果表明，胚萌发后上调表达的蛋白质数量范围仅为14-25个，而同组样品下调表达的蛋白质数量是上调数量的10倍左右；通过生物信息学分析发现，同组样品中上述两类数据的GO功能分类基本一致，即参与的生物过程主要是代谢过程，所在位置主要是细胞和细胞器，分子功能主要是结合和催化活性；结合上述相关数据的关联分析，筛选出了10个与油棕种子萌发相关的候选基因，并通过转录表达分析，初步定位了3个参与油棕种子萌发调控的重要基因。相关结果可以为进一步探索油棕种子萌发调控机理以及休眠破除技术研究提供参考。