玉米ZmMYB59转录因子参与调控种子顶土萌发的分子机制研究

Deep-sowing on the basis of the moisture in the soil is the traditional sowing method for drought resistance in maize production because seeds can absorb water in the deep soil layer to germinate. Since the existing varieties have poor germinability from the soil, it is difficult for seeds to germinate from the soil so that seedingless ridges often occur. Improving drought-tolerant varieties via the usility of the genes related to seed germinability from the soil is a new approach to resist heavy drought. However, there are no reports on the functional studies of these genes. Our previous studies demonstrated that the expression of many MYB family genes was down-regulated by deep seeding. In MYB transcription factors, ZmMYB59 had the highest folds of down-regulated expression. Therefore, we speculate that ZmMYB59 may play a negative regulatory role in the process of seed germination from the soil. On the basis of the cloning of ZmMYB59 and its promoter, the function and the model of action of ZmMYB59 involved in regulation of seed germinability from the soil will be revealed from the following three aspects.Firstly, the temporal and spatial expression patterns of ZmMYB59 and its promoter are analyzed. Secondly, the functions of germinability from the soil of seeds which ZmMYB59 is transformed into by over-expression and knock-out are characterized. Thirdly, the signal transduction pathways of ZmMYB59 are analyzed based on protein and RNA levels.Our study will lay the theoretical foundation for the eluciation of molecular mechanisms on seed germinability from the soil regulated by MYB transciption factors, and provide the key genes for maize breeding of drought-tolerant varieties with high germinability from the soil.

利用土壤深层水分的探墒深播是玉米生产中传统的抗旱播种方法。现有品种种子顶土能力差，深播很难出苗，缺苗断垄现象严重。利用种子顶土萌发相关基因改良现有耐旱品种是应对当前严峻旱情的新途径。但迄今与玉米种子顶土萌发相关基因的功能研究未见报道。我们前期研究发现，大量MYB家族基因受深播诱导表达下调，其中ZmMYB59基因表达下调倍数最高，推测它在玉米种子顶土萌发过程中起负调控作用。本项目在克隆ZmMYB59基因及其启动子序列的基础上，拟通过研究该基因和启动子的时空表达模式，利用超表达和基因敲除技术获得的转基因种子进行顶土功能鉴定，基于蛋白和RNA水平上的信号转导分析，揭示ZmMYB59基因参与调控玉米种子顶土萌发的功能和作用模式。本项目将为深入探讨MYB转录因子调控种子顶土萌发的分子机理奠定理论基础，同时为选育顶土能力强的耐旱玉米新品种提供关键基因。

为揭示ZmMYB59基因参与调控玉米种子顶土萌发的功能和作用模式，本项目从ZmMYB59基因及启动子的时空表达模式分析，ZmMYB59转基因功能鉴定，ZmMYB59转录因子的信号转导途径研究等3个方面开展工作，已完成项目计划书中规定的各项研究内容，具体如下：（1）成功克隆到ZmMYB59基因，基因全长1072bp，cDNA的长度为687bp，编码228个氨基酸，含有2个SANT结合域，为典型的R2R3-MYB转录因子。（2）应用本实验室编写的perl程序取出了该基因起始密码子前2000bp的序列，通过转基因GUS染色表明，该基因的启动子没有组织特异性，同时说明启动子前1k序列为其发挥正常调控功能所必须；将融合表达载体35S::ZmMYB59-EYFP转化水稻原生质体在激光共聚焦显微镜观察发现，ZmMYB59蛋白定位于细胞核中。（3）荧光定量PCR分析结果表明，不同顶土能力的玉米自交系胚或幼苗中ZmMYB59基因表达水平在播种2天或4天后均随着播深的增加而显著降低，而且中胚轴中该基因的表达量在播种6天或8天后亦随着播深的增加而降低，此外经GA3处理的种子在萌发3d后表达量降低。（4）ZmMYB59基因过表达后，T2代烟草、水稻、玉米种子在不同条件下的发芽能力均低于野生型，但其水稻中的同源基因OsMYBAS1敲除后发芽率低于野生型。（5）酵母单杂结果表明，该基因表达可能受到PEP羧化酶、GADPH、GDP-L-半乳糖磷酸化酶、氧增强蛋白等基因的上游调控，同时可能与转录调节因子SLK2、转录因子MYB38和NAC111等蛋白之间存在互作调控。（6）RNA-seq结果表明，20cm播深条件下ZmMYB59基因过表达不会改变种子响应赤霉素的信号转导网络，但能够迅速调动响应生长素相关基因和胁迫响应基因表达，进而促进细胞生长和种子萌发。以上结果表明，ZmMYB59基因在种子顶土萌发过程中发挥负调控作用，并与赤霉素和生长素信号途径有关，本项目对深入探讨MYB转录因子调控种子顶土萌发的分子机理奠定了坚实的基础。在本项目的资助下，已发表论文9篇（其中SCI收录4篇，后续还将发表论文），授权国家发明专利1项，主编学术专著1部，晋升正高和副高职称各1人，培养研究生3名，参加国际国内相关学术会议6次。超额完成计划书规定的各项指标。