玉米种子LEA蛋白积累机制的组学解析

Fast and high desiccation tolerance of maize seeds is vital to the mechanized harvesting of maize production. During maize seed maturation and desiccation, abscisic acid (ABA) promotes the formation of seed desiccation tolerance by regulating various protein syntheses, e.g. late embryogenesis abundant (LEA) proteins. In previous research, the applicant found that the accumulation patterns of LEA proteins in maize mutant vp5 seeds were much lower than that in wild-type Vp5. The mutant vp5 seeds contain much lower ABA amounts and display a slower desiccation during maturation compared to Vp5 seeds. However, it is largely unclear regarding the mechanism underlying the accumulation of LEA proteins regulated by ABA. In this proposed project, with vp5 and Vp5 seeds (embryos) at different stages as experimental materials, we will 1) analyze the dynamic changes of moisture and ABA content and their correlation during seed maturation; 2) find more LEA proteins/genes induced by ABA in seeds on the proteome and transcriptome levels; 3) determine the moisture and ABA thresholds for inducing the initiation of the synthesis of LEA proteins/genes; 4) study the change of LEA genes/proteins with the presence of exogenous ABA or its inhibitor; 5) determine the subcellular localization of these LEA proteins by immune electron microscopy. The expected results will play important roles in resolving the mechanism underlying the accumulation of LEA proteins regulated by ABA. The realization of this project will provide theoretical and applied basis for molecular breeding of maize cultivars with fast and high tolerance of seed dehydration.

种子脱水快、耐脱水是宜机收玉米品种的关键特性。在种子成熟过程中,ABA通过调控胁迫响应蛋白合成,促进种子脱水耐性形成。申请人发现,在玉米ABA缺失突变体vp5(脱水慢)种子中,多种LEA蛋白含量明显比野生型Vp5(脱水正常)种子中低,说明LEA蛋白的积累受ABA调控。但ABA调控LEA蛋白积累的机制并不十分清楚。本项目以vp5、Vp5种子为材料,分析种子发育过程中ABA含量的动态变化和脱水耐性获得的进程;在转录组和蛋白质组水平上系统研究成熟过程中LEA mRNA和蛋白的变化规律,分析LEA mRNA起始表达和LEA蛋白起始合成时ABA的阈值;通过离体、活体实验,验证ABA的效应;鉴定与种子脱水耐性相关的关键LEA蛋白,研究其组织、亚细胞定位,分析其行使功能的场所和转运途径。预期结果对于解析LEA蛋白积累的分子机制具有重要作用,为通过分子调控LEA蛋白合成培育耐脱水玉米品种提供依据。

种子的脱水耐性是玉米种子贮藏特性的一个重要依据。在种子成熟过程中，ABA通过调控胁迫响应蛋白合成,促进种子脱水耐性形成。前期研究发现,在玉米ABA缺失突变体vp5种子中，多种LEA蛋白含量明显比野生型Vp5种子中低，说明LEA蛋白的积累受ABA调控。本项目以vp5、Vp5不同发育时期的种子为材料，分析了种胚中ABA含量的动态变化及其与种子获得脱水耐性之间的关系；分析了种胚发育后期的蛋白质组和转录组变化，并构建了ABA与种子发育过程中的基因共表达网络；重点分析了LEA蛋白EMB564的组织定位及与玉米种子获得脱水耐性的关系；分析了玉米种子成熟脱水过程中miRNAs表达谱变化。发现授粉25天后Vp5种子逐渐获得脱水耐性，且随着发育进程，脱水耐性逐渐增强，而vp5种子获得的脱水耐性较弱；Vp5、vp5种子发育早期，两种材料种胚中ABA含量差异不大，在种子发育后期ABA含量差异明显：早期含量较高，之后呈下降趋势；在授粉29天后，Vp5和vp5种胚中ABA含量差异明显，vp5中继续下降，而Vp5中快速上升，暗示种胚发育早期ABA来自于母株提供，在发育后期种胚中ABA含量主要来自于籽粒自身合成。Vp5和vp5种胚在发育过程中的转录组和蛋白质组存在显著差异，其中emb564 mRNA (lea基因)从授粉后15天开始少量表达，之后开始大量表达；EMB564蛋白在授粉后20天的种胚中均未检测到，而在发育后期（授粉后25~40天）的种胚中均高丰度表达；EMB564蛋白在胚芽鞘中含量最高，在种子萌发32h后EMB564蛋白的含量大幅降低。上述结果暗示EMB564蛋白的表达不受ABA的调控，但与种子获得脱水耐性密切相关，为进一步阐明LEA蛋白的积累机制提供了新线索。另外，有研究表明miRNAs参与调控LEA蛋白的积累，在Vp5和vp5不同发育时期的种胚中，表达的miRNAs存在显著差异，但其是否参与调控相关LEA蛋白的积累还有待进一步挖掘和验证。 .本项目已发表SCI论文3篇，其中影响因子IF>3的2篇，待发表相关SCI论文2篇。培养硕士生已毕业1名，预毕业1名。