高羊茅热激转录因子热胁迫响应机制研究

Tall fescue (Festuca arundinacea) is one of the major species of cool-season forages and turf grasses. They are used widely in warm temperate to subtropical regions all over the world. With breeding improvement and selection over the years, the grass has become higher tolerance to various abiotic stresses. Even so, heat stress still restricts its growth and development, which seriously affects the yield of forages and the quality of lawn. Therefore, the heat-acclimate mechanism of tall fescue need to be further learnt. Present studies in model plant and main crop have showed that heat shock factor (HSF) is the master regulator in the transcription level and plays decisive roles in plant responding to heat stress and acquiring the thermotolerance. .Analysis the of gene differential expression in tall fescue transcriptome showed many HSF genes responded to heat stress. High temperature mainly induced subfamily A and B but repressed subfamily C. Therefore, this project focused on the four heat shock factors which was closely related to the thermotolerance of tall fescue and came from different subfamily. The relationship between the expression of HSFs gene and DNA methylation is explored by gene-specific methylation analysis based on polymerase chain reaction (PCR). Transcriptional activation activities of different HSFs are verified by yeast one-hybrid. The subcellular localization of different HSFs are observed in leaves of transgenic plant under high and normal temperature with laser scanning confocal microscopy. And the heat tolerance of these transgenic plants is also tested in physiological level. The aim of the project is to have an insight into the molecular and cellular mechanism of HSFs in tall fescue responding to heat stress, and be clear the effect of different HSFs on the thermotolerance of tall fescue..The results of this work will help further expound the molecular mechanism of heat tolerance in tall fescue, and provide new stress-tolerance genes for oriented cultivation of the excellent forage and turf grass varieties.

高羊茅是广泛应用于我国畜牧和草坪产业的一种冷季型草，夏季热害是其适应性的首要限制因子。耐热基因的挖掘和研究是耐热调控和育种的基础。在高羊茅（Festuca arundinacea）热胁迫转录组差异表达基因分析中发现热激转录因子（heat shock factor, HSF）与耐热性的获得密切相关。因此，本项目选择4个不同亚族的HSFs为研究对象，通过特异位点甲基化检测研究热胁迫下HSFs基因表达与其DNA甲基化的关系；利用酵母单杂交检验HSFs的转录激活活性；将HSFs基因分别与绿色荧光蛋白基因融合表达，观察热胁迫前后转基因植株叶片中HSFs亚细胞定位的变化，并检测转基因植株的耐热性。从分子和细胞水平揭示高羊茅HSFs在热响应过程中的作用机制，明确HSFs对高羊茅耐热性的影响。研究结果将为有助于进一步阐明高羊茅耐热分子机理，并为精确育种提供抗逆基因新材料。

高羊茅（Festuca arundinacea）是广泛应用于我国畜牧和草坪产业的一种冷季型草，夏季热害是其适应性的首要限制因子。耐热基因的挖掘和研究是耐热调控和育种的基础。本项目在整体分析高羊茅热胁迫差异基因表达的基础上，选取其中与耐热性密切相关的调控基因—热激转录因子（heat shock factor, HSF）为研究对象，利用亚细胞定位、酵母双杂交、转基因功能验证、特异位点甲基化检测等现代生物技术，研究了不同亚族高羊茅热激转录因子（HSF）基因响应于热胁迫并使植株耐热性提高的分子和细胞机制。主要成果如下：克隆到4个高羊茅热激转录因子基因HsfA2c、HsfA9、HsfB2c和HsfC2a；确定了高羊茅热激转录因子HsfA2c、HsfA9、HsfB2C和HsfC2a的亚细胞定位，都定位于细胞核中；确定了高羊茅热激转录因子HsfA2c、HsfA9、HsfB2C和HsfC2a的自激活活性。HsfA9和HsfA2c蛋白无自激活活性；但是HsfB2c和HsfC2a蛋白有自激活活性；初步验证了HsfA2c、HsfA9和HsfB2C可以提高高羊茅的耐热性；但是在高羊茅HSFs响应于热胁迫的分子机制中表观遗传调控不是其主要的调控方式。本项目在高羊茅耐热机制方面取得了一定的研究进展，研究结果从分子和细胞水平揭示高羊茅HSFs在热响应过程中的作用机制，明确了HSFs对高羊茅耐热性的影响。本项目有助于进一步阐明高羊茅耐热分子机理，并提供抗逆基因新材料。