低氮胁迫下杨树根系构型的miRNA调控机制

miRNA plays a vital role in the regulation of plant response to low nitrogen stress. It is highly necessary to investigate the characteristics of the response of poplar under low nitrogen environment and elucidate the genetic regulation mechanism, which is significant for the mechanism of the adaption of trees to low nitrogen and tree breeding for efficient utilization of nitrogen. In our previous research, low nitrogen stress related miRNAs and the target genes have been analyzed and identified. Furthermore, the different responses of poplar full-sib family to low nitrogen stress have been investigated and clones with different responses were identified. Based on these researches, the characteristics of roots architecture and physiological and biochemical changes of different poplar clones in response to low nitrogen stress will be further investigated. The differential expressions of miRNAs and the corresponding target genes are thereafter analyzed to identify the different expression among different poplar clones. The association between the different expression of miRNAs and the different responses of poplar is further established. For the responsive miRNAs and targets, the specific promoters induced by low nitrogen are further analyzed. Four to six expression vectors are constructed and the localization or induced expression of promoters in different tissues or treatment time points is analyzed. The core elements of promoters are identified to reveal the regulatory mechanism of poplar miRNAs in response to low nitrogen stress. To further identify the function of regulatory miRNAs, two to three over-expression or suppression expression vectors are constructed and the transformation is done in poplar. The changes of roots architecture, physiological and biochemical responses and metabolic content under low nitrogen will be investigated and differential expression of miRNAs or target genes will be detected in the positive transgenic poplars. This research will provide new insights for the genetic mechanism of the adaption of trees in response to low nitrogen stress and critical candidate genes to tree breeding for efficient utilization of nutrition.

miRNA在调控植物对低氮胁迫的适应性上起着重要作用。开展miRNA调控杨树适应低氮胁迫遗传机制的研究，对于阐明林木适应氮胁迫遗传机理并加速林木营养高效育种具有重要理论意义和应用价值。本申请在前期开展杨树受低氮胁迫相关miRNA及靶基因鉴定及杨树全同胞家系耐低氮胁迫差异性鉴定的基础上，分析低氮处理不同时间段杨树根系构型及生理生化变化，鉴定miRNA、靶基因的差异表达变化，建立差异表达miRNA与杨树对氮胁迫不同耐受性的关联；对差异表达变化的miRNA、靶基因，鉴定其启动子并进行缺失分析和组织表达，解析启动子关键位点、核心元件及表达特征；构建2-3个关键miRNA、靶基因的过表达/抑制表达载体，在杨树中进行遗传转化，分析转基因植株根系构型变化及生理生化变化，并对转基因植株进行氮胁迫耐性测定。通过上述研究，为林木适应低氮胁迫分子机理的研究提供新思考，为林木营养高效育种提供重要的候选基因。

miRNA是一类重要的非编码小分子RNA，在调控植物对低氮胁迫的适应性上起着重要作用。开展miRNA调控杨树适应低氮胁迫遗传机制的研究，对于阐明林木适应氮胁迫遗传机理并加速林木营养高效育种具有重要理论意义和应用价值。为了鉴定miRNA、靶基因的差异表达变化，解析低氮处理不同时间段杨树表型及生理生化变化，建立差异表达miRNA与杨树对氮胁迫不同耐受性的关联，本研究在课题组前期研究的基础上，对杨树进行不同时间段的低氮胁迫处理，利用实时定量PCR技术研究毛白杨在低氮胁迫后10种miRNA的差异表达模式。结果显示，miRNA的表达在低氮胁迫下有显著变化，部分miRNA的表达呈现动态反应。在低氮胁迫下，miRNA的表达呈现四种模式，大部分miRNA的表达在处理1 d后受到显著抑制。借助生物信息学手段，进一步鉴定到10个miRNA的靶基因，功能注释表明靶基因共分为6大类，降解组实验进一步验证了miR159a对靶基因的切割。上述结果表明，miRNA在杨树对低氮营养的胁迫响应中发挥着重要调控作用。获得了131个miRNA家族的242个miRNA成员对应的3024个靶基因，miR162的靶基因编码ABC转运蛋白，miR393作用于靶基因KAT2，可调节Na+和K+动态平衡，miR399的靶基因PIF3编码光敏色素互作因子PIFs蛋白。开展了低氮处理的杨树转录谱分析，鉴定出1, 561个下调和1, 101个上调的差异表达基因(DEGs)，有20个DEGs参与了氮代谢途径，与碳水化合物代谢有关的四个途径也得到了富集。获得了杨树miR171c上游1, 631bp的启动子序列，并分析了其调控元件组成。成功克隆了长度为370 bp的毛白杨miR171c前体并构建了其过表达载体，通过农杆菌转化法对银腺杨无性系84K进行了遗传转化，鉴定得到5株miR171c过表达阳性植株。开展了杨树在低氮胁迫后的表型及生理生化测定，低氮胁迫下碳-氮的合成存在关联调控，以调整杨树的生理生化变化进而调控杨树低氮瘠薄环境的适应性。本研究为进一步探讨杨树miRNA在响应低氮胁迫中的功能提供了一定的理论线索，有利于揭示杨树响应低氮胁迫的miRNA调控机制，同时为树木营养高效育种的分子改良提供了候选基因。