玉米渍害胁迫诱导microR528介导的基因表达调控网络的研究

Maize is the one of the world’s most important crop for food, feed and energy. And it is also the largest crop for food in China. As the waterlogging and follding accored more oftern during this decade, waterlogging has become to the second most important abiotic stress in restricting the development of maize production. In eukaryotic genome, there are a large number of non-conding RNAs, especially miRNA, which directly involved in the regulation of the developmental and stress response gene expression in the transcriptional and post-transcriptional level..In this study,the roots from 368 association mapping population were used for analysis. And selected the miR528, which is expecially highly represented in the crown root and in responding to waterlogging stress, as candidate miRNA for research. By association analyzing the miR528 candidate gene with its expression and woterlogging resistant traits in those maize inbred lines. And screening the newly constructed transcription factor library, which invloved in responding to waterlogging stress, to validated the transcription factor binding site on the miR528 promoter. Moreover, qPCR and RACE are used to analyze the miR528 target genes in order to construct miR528 mediated gene regulatory network and characterize the roles of miR528 in responding to waterlogging in maize root cell, which will give a new sight of the miR528 regulation mechanism under waterlogging stress and help on the marker-assisted selection of the waterlogging resistance gene and breed excellent waterlogging.resistance inbred lines.

玉米是全世界最重要的粮食、饲料和能源作物之一,也是我国第一大粮食作物。随着全球气候的极端变化,涝渍的发生频率在逐年递增,并已逐渐成为继干旱之后威胁玉米生产的第二大非生物逆境胁迫。已有研究证明miRNA在转录和转录后水平上,直接参与发育基因和对非生物逆境胁迫应答基因的表达调控。本研究选用368份关联群体自交系为实验试材料,并选取在冠根特异表达并对渍害逆境响应的miR528作为研究对象。拟建立miR528候选区段与miR528表达以及耐渍表型的关联关系,并筛选酵母单杂交文库,验证参与调控miR528表达的转录因子;利用QPCR和RACE分析并证实渍害胁迫下玉米根系中miR528靶基因的表达,建立miR528介导的基因调控网络并阐明其与渍害胁迫条件下冠根发育间的关系,为玉米耐渍性遗传改良提供新的基因资源和策略。

MicroRNA528是单子叶植物中高度保守的miRNA之一。许多研究证明，miR528能够响应多种非生物胁迫，而我们之前的研究也表明，zma-miR528可能是参与根系发育和响应淹水胁迫的一种关键miRNA。然而，玉米自交系间Zma-miR528表达差异对淹水胁迫的响应机制尚不清楚。在本研究中，我们克隆并鉴定了许多玉米自交系的Zma-miR528启动子区，这些自交系由来自热带和温带地区的368个品种组成。利用不同群体的玉米，研究了缺氧胁迫下zma-miR528启动子核酸变异与成熟zma-miR528表达差异间的关系。. 结果表明，Zma-MIR528启动子的遗传变异与苗期成熟Zma-miR528的表达差异间具有显著的相关性。低氧胁迫下，调控zma-miR528差异表达的相关位点主要位于zma-miR528a的启动子区上。启动子分析表明，在Zma-MIR528启动子中，存在许多重要的顺式作用元件，如Dof、MYB、ARF等。此外，我们还研究了低温、ABA、盐胁迫等短时间（8h）处理下玉米根成熟zma-miR528的表达情况。结果表明，不同胁迫条件下其表达显著且各处理间表达模式不同。由此可见，MIR528在玉米逆境胁迫中起着重要作用，成熟Zma-miR528的表达模式有助于玉米适应环境的变化，表明调控Zma-miR528在改善植物非生物逆境抗性方面具有较高的研究价值。