沟叶结缕草ZmDUF1644基因的耐盐转录调控机制研究

Zoysia matrella, a perennial halophyte, is an excellent plant material for improvement and utilization of saline-alkali. Due to the lack of research on the molecular mechanism of salt tolerance transcriptional regulation, the development and application of Zoysia matrella salt tolerance has been greatly restricted. Previous research have found that DUF1644 is a plant-specific DUF protein that plays an important role in response to salt stress and can significantly improve the salt tolerance of transgenic rice. However, its molecular mechanism is still unclear and has not been reported in perennial halophytes. In previous study, we identified a ZmDUF1644 gene from Zoysia matrella. The salt tolerance of ZmDUF1644 transgenic Arabidopsis plants was significantly improved. However, the salt-tolerant transcriptional mechanism of ZmDUF1644 gene is still unknown, and Studies have found that the salt-tolerant transcriptional regulation mechanism of halophytes tend to different from non-halophytes. This project mainly uses transgenic technology and molecular biology methods to study two major issues: One is to identify the core component of the ZmDUF1644 promoter and define the salt tolerance function of ZmDUF1644 in Zoysia matrella; The other is to screen and validate the transcription factor that interacts with the core elements and to elucidate the molecular mechanism of transcriptional regulation of ZmDUF1644. Execution of this research can not only enrich the understanding he molecular mechanism of halophyte plants, but also provide excellent genetic resources for salt tolerance breeding in other turfgrass.

沟叶结缕草是多年生盐生植物，是改良和利用盐碱地的优异植物材料。由于缺乏耐盐转录调控分子机制的研究，极大地限制了其耐盐性的开发和应用。前人研究发现DUF1644是植物特有的DUF蛋白，在响应盐胁迫过程中发挥着重要作用，可显著提高转基因水稻的耐盐性。但其分子机制尚不明确，且在多年生盐生植物中未见报道。前期，我们从沟叶结缕草中筛选到一个ZmDUF1644基因，可显著提高转基因拟南芥耐盐性。然而，其转录调控机制仍然未知，且有研究发现盐生植物耐盐转录调控机制往往不同于非盐生植物。本项目主要利用转基因技术和分子生物学手段，研究两个主要问题：一识别ZmDUF1644启动子核心元件，并在沟叶结缕草中明确ZmDUF1644的耐盐性功能；二筛选验证与核心元件互作的转录因子，并阐明ZmDUF1644转录调控分子机制。本项目的实施不仅将深化对盐生植物耐盐分子机制的理解，同时也为其他草坪草耐盐育种提供优异基因资源。

沟叶结缕草作为一种多年生盐生植物，对于盐碱地改良与滩涂绿化具有重要的意义。深入解析沟叶结缕草耐盐基因的转录调控机制，不仅能够为植物抗逆功能研究提供理论基础，而且可为草业抗性育种提供关键的候选基因和资源材料。在植物中，DUF1644家族基因在应答盐胁迫方面发挥着重要的作用，但其上游调转录调控机制仍不清楚。本研究对ZmDUF1644进行了系统的生物信息学分析，发现ZmDUF1644蛋白含有一个典型的DUF1644结构域和C2H2型的锌指结构，且与水稻OsSDIP361同属于GroupII类DUF1644蛋白。qRT-PCR和亚细胞定位结果显示ZmDUF1644基因是一个组成型表达基因，主要定位于细胞质，且在NaCl处理24h表达量达到最高。盐胁迫下，在拟南芥和水稻中过表达ZmDUF1644基因后，植株生长较野生型表现出更优的生长状态。特别是拟南芥中，转基植株的生物量显著高于野生型，并且呈现出更低的电解质渗漏率和更高的叶片相对含水量，此外，叶片中活性氧积累减少，离子含量测定也发现转基因拟南芥叶片和根系中具有较高的K+/Na+。在此基础上，通过酵母单杂筛选获得了ZmDUF1644上游调控因子ZmROC1和ZmPTF，体内外实验均表明ZmROC1和ZmPTF与ZmDUF1644启动子均真实结合，且结合位置分别在ATG上游801-1000序列以及ATG上游1000-1199序列之间。通过EMSA实验，进一步明确了ZmROC1调控ZmDUF1644基因上游启动子的核心元件为“GCCACCT”。以上结果表明ZmDUF1644基因在响应盐胁迫过程中具有极其重要的作用，且ZmROC1通过与核心序列“GCCACCT”结合，调控ZmDUF1644基因响应盐胁迫。