ZmNAC调控苗期玉米应答低温冷害的分子机制研究

NAC transcriptional factor is an important transcription factor (TF) in regulation of gene expression of plant development and stress tolerance. In previous work, we have obtained 30 cold up-regualated ZmNACs belonging to 12 subfamily by comparing the leaf-transcriptomes of maize under normal condition with cold treatment. ZmNACs which low temperature responsive element are highly enriched in the promoters were chosen for further study. First, the cold-resistant function will be identified in ZmNACs overexpression arabidopsis and restore mutations. Then the basic characteristics of transcriptional factors including expression patterns of ZmNACs under stresses, subcellular localization, trans-activation activity and dimer-interaction will be further analyzed. Lastly, in order to reveal the molecular mechanism of ZmNACs in regulation of chilling injury at maize seedling stage, ChIP-Seq, yeast one-hybrid, transient coexpression and EMSA will be used to systematically research the key motif recognized by ZmNAC to regulate gene expression during cold-promoted stress response. This study will construct a molecular regulatory working model about the function of ZmNAC transcriptional factors in response to cold stress, which is helpful to further understanding the mechanism of TFs in regulation of cold tolerance in corn seedling, and will also provide the new candidate genes for cold-tolerant crop breeding.

NAC转录因子是一类调控植物发育及抗逆基因表达的重要转录因子。前期，通过比较正常和冷胁迫处理下玉米叶组织转录组，我们已经获得了30条冷上调ZmNACs，它们分属于12个亚族。选择启动子中富含低温响应元件的ZmNACs进一步研究。首先利用过表达拟南芥和恢复突变体分析ZmNACs的耐冷功能；然后对NAC逆境下的表达模式、亚细胞定位、转录激活活性以及二聚作用等转录因子基本特性进行分析；最后，为揭示ZmNAC调控苗期玉米应答低温冷害的分子机制，拟利用ChIP-Seq、酵母单杂交、瞬时共转化和EMSA等技术对ZmNAC在冷胁迫下调控基因表达所识别的关键元件进行系统研究。本研究将构建ZmNAC应答冷胁迫的分子调控模型，这有助于深入理解转录因子调控的玉米幼苗耐冷机制，并进一步为农作物耐冷育种提供新的候选基因。

低温严重制约作物生产和农业发展。前期研究中，我们已经获得了30条低温下上调的ZmNACs基因。本项目选取两条低温响应基因ZmNAC12和ZmNAC62进一步研究。ZmNAC12和 ZmNAC62蛋白都定位在细胞核，它们的单体都没有转录自激活活性。ZmNAC12或ZmNAC62在拟南芥中的过表达能够提高转基因植物对低温的耐受性。低温胁迫条件下，ZmNAC12和ZmNAC62均可以增加SOD和POD基因的表达，进而提高活性氧（ROS）清除能力。ZmNAC12可以诱导脯氨酸合成以及叶绿素合成相关基因（P5CS1、P5CS1、HEMA1、HEMB1和CHLH）的表达，从而引起脯氨酸和叶绿素含量的增加。此外，ZmNAC12可以显著诱导冷胁迫标志基因CBF1和CBF3的表达。相反，ZmNAC12抑制花青素合成相关基因F3H和ANS的表达，导致花青素含量的减少。酵母单杂交实验表明ZmNAC12和ZmNAC62可以特异性的结合逆境相关基因启动子中的NACRS顺式作用元件，表明ZmNAC12和 ZmNAC62通过识别NACRS进而参与调控这些逆境相关基因的转录。本研究鉴定了ZmNACs在低温胁迫反应中的功能，初步阐明NAC转录因子所介导的植物应答低温胁迫反应的分子基础。这些发现将拓宽我们对低温应激反应调控网络的认识，为进一步研究低温胁迫响应的分子机制和耐低温作物的分子育种提供重要线索。