根特异低氮响应MYB基因调控甘蔗氮利用效率的分子机制研究

Nitrogen (N) is a major essential element for sugarcane taken up from the soil, which has significant impacts on sugarcane growth and development. It is often the most limiting factor in sugarcane production. Therefore, over-fertilization of nitrogen in present sugarcane production in China is very common. The low nitrogen use efficiency (NUE) and a vast amount of nitrogen fertilizers poured into the fields increase both the cost and the risk for environmental contamination. In this context, breeding for sugarcane nitrogen efficient cultivars became an effective way both to improve the NUE and to reduce the nitrogen fertilizer application. The molecular mechanisms of NUE research are the basis and the key to obtain sugarcane cultivars with high nitrogen use efficiency. Four root-specific low-nitrogen responsive MYB transcription factor genes, termed as ScRSLNR-MYBs, have been identified in our previous research. In further transient expression assay and nitrogen metabolism related enzyme activities test in tobacco, these genes had been preliminarily verified to involve in the regulation of nitrogen metabolism. In this proposal, four ScRSLNR-MYB genes will firstly be over-expressed in the root of model-plant foxtail millet (Setaria italic)【belong to C4 crop in Gramineae, similar to sugarcane】respectively, and then NUE, phenotype, physiologic and biochemical indexes and the expression of nitrogen metabolism-related genes will be evaluated in foxtail millet transgenic lines compared with non-transgenic line. Subsequently, 1~2 ScRSLNR-MYBs will be selected as key candidate genes according to their performance in foxtail millet transgenic lines (positive or negative role in NUE). After that, their transgenic sugarcane lines will be produced by over-expressed or knock-down (RNAi via siPools technology) and similar assay will be performed.Combination the experimental data of the above and the interactions of ScRSLNR-MYBs and their target genes or proteins, we propose to elucidate the molecular mechanisms of regulation in sugarcane nitrogen use efficiency via root-specific low-nitrogen responsive MYB.

氮素对甘蔗生长发育与产量形成具有重要意义。我国甘蔗生产普遍过施氮肥，氮利用率偏低，培育氮高效品种是提高氮利用率、减少施氮量的有效途径，而氮利用的分子机制研究是培育氮高效品种的前提基础与关键。项目前期从甘蔗中挖掘到4个根特异表达且低氮胁迫响应敏感的MYB转录因子（ScRSLNR-MYB），烟草瞬时表达及氮代谢相关酶活测定初步证明它们参与氮代谢调控。鉴于甘蔗转化效率低，项目拟先在模式植物谷子（与甘蔗同为禾本科C4作物）根系中定向过表达上述基因，评价相应转基因谷子的相关性状，快速筛选出1-2个氮代谢调控关键基因。根据关键基因特性（提高或抑制谷子氮利用效率），在甘蔗主栽品种ROC22中，采用过表达或基于siPools的RNAi技术，创制转基因系，评价其在低氮条件下的表型、氮利用效率、相关生理生化与基因表达情况，结合互作蛋白和靶基因筛选及验证，揭示ScRSLNR-MYB调控甘蔗氮利用效率的分子机制。

甘蔗（Saccharum spp. hybrid）是世界上最主要的糖料作物和生物能源作物。我国甘蔗生产上普遍过量施用氮肥，然而氮利用率偏低，增加了生产成本，还影响环境。调控甘蔗氮素利用的关键转录因子的功能鉴定与分子机制研究是培育甘蔗氮高效品种的前提与关键。项目组首先从转录调控的角度解析甘蔗低氮高效的分子机制，通过对两个基因型（低氮耐受型和低氮敏感型）甘蔗在低氮胁迫下的表型、生理与差异表达基因集进行关联分析，富集和构建影响甘蔗氮高效利用的转录调控网络，并从中确定和克隆候选MYB转录因子。项目组对其中的4个关键候选MYB转录因子进行生物信息学、亚细胞定位和表达模式分析，通过蛋白互作和（或）启动子绑定分析揭示它们的调控关系。同时，构建表达载体并转化拟南芥和甘蔗进行功能验证，获得了过表达ScMYB52和ScMYB11的甘蔗转基因系和干扰ScMYB11表达的甘蔗转基因系。综上，本项目挖掘到4个参与调控甘蔗氮素利用的MYB转录因子，初步明确了其在调控植株利用氮素过程的作用和互作因子，为氮高效甘蔗分子育种提供了理论基础、基因元件和转基因甘蔗材料。