水稻膜结合转录因子OsNTL3调控高温等逆境胁迫响应的分子机理

Abiotic stresses are great threats to agriculture production. Reducing crop yield loss and quality penalty by stress resistance improvement is a fundamental guarantee for sustainable agriculture. Membrane-associated Transcription Factors(MTFs) are a class of transcriptional regulators with hydrophobic transmembrane domains, which are normally dormant because of the membrane anchors. Therefore, they not only have functions in gene expression regulation, but also play roles in sensing the signals. Growing evidences show that MTFs play important roles in responses to environmental stresses in plants. Our previous studies discovered one rice membrane-associated NAC transcription factor OsNTL3, which is induced by protein misfolding inducers TM and AZC. To further investigate the functional characteristics and its molecular mechanisms of OsNTL3 in rice response to abiotic stresses, we intentionally carry out studies at molecular, biochemical and genetical levels. The research aspects include: OsNTL3 expression pattern during development and in response to abiotic stresses; OsNTL3 subcellular localization and organelle-to-organelle translocation; interaction partners screened by yeast two hybrid system; downstream genes under environmental stresses obtained by using loss-of-function and gain-of-function rice mutants created by CRISPR/CAS9 system. These results will reveal the biological functions and signal transduction pathway of OsNTL3, and may provide new gene and method for genetic improving rice resistance to abiotic stresses. The findings may also increase our understanding of the functions and activation mechanisms of MTFs in plants.

逆境胁迫会引起水稻等作物产量减少，品质降低。提高作物的抗逆性，稳定农产品产量和品质是关系社会可持续发展的重要保证。膜结合转录因子是一类具有疏水跨膜域的转录因子，拥有在不同亚细胞器间转移和活化的特性，因而具有感应信号与调控基因表达的双重功能。本课题组在前期研究中发现了一个受蛋白错误折叠诱导剂TM和AZC诱导表达的水稻膜结合NAC转录因子OsNTL3。拟进一步研究OsNTL3的表达模式及对逆境胁迫的响应模式、亚细胞转移等生物学特征；用酵母双杂交筛选互作蛋白；用CRISPR/CAS9系统创制功能缺失和功能获得型材料，研究OsNTL3在逆境胁迫应答中的功能及其调控的下游基因。通过本项目的实施，可揭示水稻OsNTL3的生物学功能和信号转导通路，并获得遗传改良水稻抗逆性的基因与方法，为深入理解植物膜结合转录因子的功能与活化机理奠定基础。

内质网是所有分泌蛋白和大部分膜蛋白合成和折叠的场所。当错误折叠蛋白积累时，细胞会启动未折叠蛋白应答途径来帮助蛋白折叠或降解错误折叠蛋白。水稻OsNTL3表达受蛋白错误折叠诱导剂TM和AZC的诱导，也响应DTT、SA和高温。启动子活性分析表明，OsNTL3在幼苗和成熟水稻各个组织中都有表达，其中在幼苗的根中和生殖器官中表达量较高。结构域分析表明，OsNTL3蛋白含有一个N端的NAC结构域和一个C端的跨膜域（TM），高温胁迫使OsNTL3活化，形成一个新的较小的蛋白。与YFP融合的OsNTL3在烟草表皮细胞中定位于内质网，与内质网marker共定位，而去除跨膜域的OsNTL3则定位于核中，暗示在高温胁迫下OsNTL3蛋白经活化后从内质网膜转移到细胞核内。运用CRISPR/CAS9技术获得在OsNTL3的NAC结构域突变和在跨膜域突变的基因定点编辑水稻材料（ntl3-1和ntl3-2），由于两种材料的阅读框移码，造成其编码的氨基酸序列提前终止或改变。转录活性分析表明，去除跨膜域的OsNTL3在酵母中具有转录活性，而氨基酸改变的NTL3-2则无转录活性，说明ntl3-2是功能缺失突变体。OsNTL3功能缺失突变体对高温更加敏感，在高温胁迫下其存活率相对于野生型大大降低。RNA-SEQ实验表明，高温胁迫诱导下显著上调的基因在野生型水稻中比OsNTL3功能缺失突变体中多，其中1001个基因在野生型中的上调幅度至少是突变体中上调幅度的1.5倍。挑选其中FPKM>10的463个基因进行GO富集分析，结果表明响应温度胁迫、响应氧化胁迫的基因比较富集。本研究揭示水稻OsNTL3的生物学功能和信号转导通路，为深入理解植物膜结合转录因子的功能和活化机制奠定基础。