基于CRISPR/Cas 9 技术解析多年生黑麦草耐盐MATE基因分子调控网络

Perennial ryegrass (Lolium perenne L.) is a typical cool season forage grass. There is 22.5 million hectare saline soil in China. Some of the coastal beach land contains more than 3.0 % salt， which limits the Popularization and Application of perennial ryegrass. Improvement of perennial ryegrass salinity tolerance by modern breeding technology is one of the most important methods for getting new germplasm. Our previous research indicated that MATE gene ( Multitrug and toxic compound extrusion protein) was up-regulated by 8 times when plant was subjected to salinity. The project will be conducted to investigate the molecular mechanism of MATE gene mediate salinity tolerance of perennial ryegrass by performing the following experiments: ①Analyzing expression of salinity tolerance of MATE gene based on the transgenic technology and CRISPR/ Cas 9; ② Identifying the upstream transcription factor of MATE gene through promoter deletion mutant and yeast one hybrid; ③ Explore MATE downstream gene through RNA-seq technology. Execution of this research can not only enrich the understanding of the molecular mechanism how MATE regulates salinity tolerance of plants, but also provide theoretical basis for molecular breeding of other salt tolerance forage grass based on MATE gene.

多年生黑麦草（Lolium perenne L.）为典型的冷季型牧草。中国重度盐碱地达2250万公顷，大面积的滨海滩涂地含盐量高达3%以上，限制了多年生黑麦草的栽培应用。通过现代育种技术改良其抗盐性，是获得黑麦草耐盐品种的重要手段。通过前期研究，我们鉴定到多年生黑麦草受盐胁迫诱导表达的MATE基因，本课题通过以下试验研究其耐盐分子机制：①基于过量表达和CRISPR/Cas 9 基因编辑技术对MATE基因进行耐盐功能分析；②采用启动子分段缺失或突变以及酵母单杂交发掘和验证MATE基因的上游转录因子；③通过过量表达和基因编辑技术，揭示MATE基因上游转录调控因子的转录活性及耐盐功能；④通过RNA-seq技术，发掘MATE下游基因。本项目的成功实施不仅有助于深化理解MATE基因调控多年生黑麦草耐盐的分子机制，也将为基于MATE基因的耐盐牧草分子育种提供重要的理论依据。

多年生黑麦草（Lolium perenne L.）为典型的冷季型牧草。中国重度盐碱地达2250万公顷，大面积的滨海滩涂地含盐量高达3%以上，限制了多年生黑麦草的栽培应用。通过现代育种技术改良其抗盐性，是获得黑麦草耐盐品种的重要手段。本研究主要获得以下研究成果：①克隆到MATE基因的全长序列并生物学信息分析；并克隆其启动子序列分析其互作元件；进行亚细胞定位，发现MATE定位于细胞膜；②根据Plantcare网站预测并分析启动子序列，发掘MATE基因的上游可能结合的转录因子，进行酵母单杂验证，最终验证有两个转录因子可能与MATE基因的启动子存在互作关系；③MATE基因转化拟南芥后，可以增强拟南芥的耐盐功能；④基于过量表达和CRISPR/Cas 9 基因编辑技术对MATE基因进行耐盐功能分析。本项目的成功实施不仅有助于深化理解MATE基因调控多年生黑麦草耐盐的分子机制，也将为基于MATE基因的耐盐牧草分子育种提供重要的理论依据。