HvmiR393介导的生长素信号调控在大麦籽粒发育和萌发中的作用机制研究

Grain development and germination are two important developmental stages which affect barley production and malt quality. Our previous studies identified HvmiR393/target pair as a novel regulator which is highly expressed during grain development and germination in barley. Phenotype observation of transgenic barley with altered miR393 levels suggested that miR393-induced target repression is involved in the regulation of seed size, weight and germination rate. This project aims to establish a precise spatio-temporal expression pattern of HvmiR393, and investigate the relationship among miR393 accumulation, target expression and auxin response. Overexpression and knockdown transgenic barley lines will be used to reveal the biological role of miR393/target module on barley grain development and germination. Expression of related gene and hormone content will be detected to study the regulation role of miR393 on auxin signaling pathway and crosstalk between auxin and ABA/GA. The project also includes metabolomics analysis of metabolities changes in seeds, measurement of seed size, weight and composition, observation of seed germination and seedling growth, so that we can explain how miR393 affects grain development and germination in barley. The mechanism of small RNA-mediated regulation of grain development and germination could provide new strategies for barley quality improvement.

籽粒发育和萌发是影响大麦产量和麦芽品质的重要因素。前期工作中我们筛选并验证了一个在籽粒发育和萌发过程中高表达的HvmiRNA393/靶基因作用模块，转基因材料的表型分析说明HvmiR393通过靶基因抑制，调控籽粒大小、重量和萌发特性。本项目拟通过原位杂交、定量qRT-PCR以及启动子报告株系的组织染色等方法，建立大麦籽粒发育和萌发过程精细的HvmiR393时空表达谱，揭示HvmiR393积累与靶基因表达及生长素反应的相关性。利用miR393过量表达和功能抑制的转基因大麦株系，通过基因表达检测和激素含量测定，研究miR393对生长素反应的调控，以及与脱落酸和赤霉素信号的互作。通过代谢组分析、种子结构和组成的测定，萌发率和幼苗生长特性的观察，探寻miR393对籽粒发育和萌发的调控功能。最后提出新的基于小分子 RNA途径调控大麦籽粒发育和萌发的分子机制，为大麦品质的遗传改良提供新思路。

籽粒发育和萌发是影响大麦产量和麦芽品质的重要因素。前期工作中我们筛选并验证了HvmiRNA393/靶基因作用模块，本项目进一步探索了miR393对大麦籽粒发育和萌发的调控功能。通过Northern blot、定量qRT-PCR以及启动子报告株系的组织染色等方法，建立了大麦籽粒发育和萌发过程精细的HvmiR393时空表达谱。利用miR393过量表达和功能抑制的转基因大麦株系，发现在种子发育及萌发过程中miR393通过负调控生长素反应信号，与ABA和GA信号互作，影响籽粒大小和重量，影响萌发对ABA的敏感性。此外，miR393介导的生长素信号途径参与主根伸长及其对酸铝胁迫的敏感性调控；miR393通过生长素反应，与气孔发育相关转录因子互作，影响气孔密度和保卫细胞长度，并通过与ABA信号互作，影响植株对干旱胁迫的耐受性。总之，发育过程中miR393的表达由内部的发育信号决定，同时受到环境因素的影响，其自身积累水平呈现时空特异性，从而进一步对其靶基因产物的精确定位做出贡献。大麦miR393通过转录后抑制靶基因TIR1/AFB的表达水平，是生长素反应的重要调控方式之一，在大麦生长发育及非生物胁迫应答反应中发挥重要调节作用。miR393/靶基因模块可为大麦品质的遗传改良提供新思路。