辽宁碱蓬NAC转录因子介导活性氧代谢途径调控植物耐盐性机制分析

盐生植物有诸多耐盐机制响应盐胁迫，保证其在盐渍环境下正常生长。NAC是高等植物特有的一类转录因子，在多种胁迫应答中发挥作用。本课题组从盐生植物辽宁碱蓬中克隆了2个NAC基因(SlNAC1/2)，转基因拟南芥盐胁迫下成活率提高，抗氧化酶活性增强，活性氧代谢相关基因上调表达。本项目拟对SlNAC1/2耐盐机制进行深入研究。ChIP-seq分析SlNAC1/2结合的启动子序列，鉴定SlNAC1/2靶位点DNA序列；检测盐胁迫下转基因拟南芥、辽宁碱蓬活性氧含量及活性氧代谢相关基因的表达；酵母单杂交、双荧光素酶分析、EMSA验证SlNAC1/2与差异表达活性氧代谢相关基因启动子的相互作用，获得转基因拟南芥、辽宁碱蓬中SlNAC1/2直接调控的活性氧代谢相关靶基因。构建“SlNACs→活性氧代谢相关基因→活性氧含量→耐盐性状”的分子调控网络。本项目将为揭示盐生植物耐盐机制、利用耐盐基因资源奠定基础。

Halophytes have many salt tolerance mechanisms to respond salt stress and to ensure the normal growth in saline environment. The plant-specific NAC (NAM, ATAF1/2, CUC2) transcription factors (TFs) play important roles in multiple stress responses. We have obtained 2 NAC TFs genes (SlNAC1/2) from the halophyte Suaeda liaotungensis. Over-expression of SlNAC1/2 improved survival ratio of transgenic Arabidopsis, enhanced the antioxidant enzyme activities, and up-regulated the reactive oxygen species(ROS)-related gene expression under salt stress. Basing on the previous work, this project will be performed on investigating the salt tolerance molecular mechanisms of SlNAC1/2 TFs. First, the SlNAC1/2 binding promoter sequence are obtained by ChIP-sequence. And the SlNAC1/2 binding sites are identified using MEME. Second, the ROS concentration and ROS-related gene expression under salt stress are detected in SlNAC1/2 transgenic Arabidopsis and Suaeda liaotungensis. The interaction between SlNAC1/2 TFs and the promoters of differentially expressed ROS-related genes are tested by yeast one hybrid, dual-luciferase reporter assay, and electrophoretic mobility shift assay. The target ROS-related genes directly regulated by SlNAC1/2 TFs are obtained. Finally, the molecular regulating network of “SlNACs → downstream ROS-related target genes → ROS concentration → salt-tolerant traits” will be constructed and it can help us understanding the salt tolerance mechanisms of NAC TFs. This study will lay a foundation for revealing the salt tolerance mechanism, and utilizing the salt tolerance gene resources in halophytes.