海滨雀稗耐盐相关K+、Na+离子平衡关键基因的功能分析

Seashore paspalum is a halophytes turfgrass with extreme salt tolerance; however, its molecular mechanism in salt tolerance is still unknown. In our preliminary investigation，several individual plants with difference in salt tolerance have been selected; The potential K+, Na+ ion balance-related genes were identified based on transcriptome sequencing data, and Agrobacterium-mediated transformation in seashore paspalum has been established. Thus the key genes associated K+, Na+ ion balance-related genes in response to salinity stress and salinity tolerance mechanism in seashore paspalum are going to be studied in this proposal. The genes showing differential expression between salinity tolerant and susceptible plants will be screened, followed by screening for plasma membrane- or tonoplast-localized protein encoding genes by using the method of cellular localization analysis. The candidate proteins showing ion channel characteristic or ion transport activity and association with salinity tolerance will be identified using a yeast expression system. On the basis of the above data, one or two key genes will be selected for functional analysis using transgenic seashore paspalum plants by overexpressing the candidate gene(s) or down-regulating expression of the candidate gene(s). Transgenic plants were generated using Agrobacterium-mediated transformation after several experimental steps including co-cultivation of embryo calli, selection of basta-resistant calli, regeneration of plantlets. Salinity tolerance and ion homeostasis will be analyzed in transgenic plants in comparison with the wild type. Nevertheless, the investigations will provide new knowledge revealing molecular mechanisms underlying salinity tolerance in seashore paspalum and help design molecular breeding strategies in genetically engineering turfgrass or other crop plants for improved salinity tolerance.

盐生植物海滨雀稗是耐盐性最强的草坪草，但其耐盐分子机理尚不清楚。前期工作已筛选出耐盐差异明显的海滨雀稗材料，利用转录本数据归纳了K+、Na+离子平衡相关基因，并已建立成熟的海滨雀稗转基因技术。在此基础上，本项目拟开展以下研究：系统分析耐盐差异明显的海滨雀稗材料间K+、Na+离子平衡相关基因响应盐胁迫的表达模式；通过亚细胞定位筛选定位于质膜或液泡膜的蛋白；利用外源酵母系统快速鉴定出具有离子转运活性并提高酵母耐盐性的候选蛋白。最终挑选出1-2个关键基因，构建其过表达载体和RNAi载体，导入愈伤组织，经筛选获得转基因再生植株。通过分析转基因株系的耐盐性及体内K+、Na+离子稳态，阐明候选基因在耐受盐胁迫中的功能及作用机制，为解析海滨雀稗耐盐机制奠定基础。本项目研究不仅具有重要科学意义，所挖掘的耐盐相关基因也可望应用于草坪草耐盐性改良，为草坪草耐盐分子育种提供依据，有潜在应用价值。

盐生植物海滨雀稗是耐盐性最强的草坪草，但其耐盐分子机理尚不清楚。本项目在利用转录本数据归纳了K+、Na+离子平衡相关基因并建立海滨雀稗转基因技术的基础上，系统分析了耐盐差异明显的海滨雀稗材料间K+、Na+离子平衡相关基因响应盐胁迫的表达模式，从中挑选出三个K+、Na+离子平衡相关基因PvHAK12, PvHAK16及PvBASS2。利用外源酵母、拟南芥系统鉴定蛋白的离子转运活性及在植物响应胁迫过程中的功能，发现这三个基因负调控植物的耐盐性。同时构建了PvBASS2过表达载体和RNAi载体，导入愈伤组织，经筛选获得转基因海滨雀稗再生植株，确定PvBASS2过表达转基因株系降低耐盐性而RNAi株系提高耐盐性，并且耐盐性的改变与PvBASS2影响光合作用过程有关。本项目研究不仅具有重要科学意义，所挖掘的盐敏感相关基因也可望应用于草坪草耐盐性改良，为草坪草耐盐分子育种提供依据，有潜在应用价值。