转录因子HSFA2与MBF1c对不同葡萄抗热性差异影响机制的研究

Grapevine is a fruit crop with important economic value and high temperature is an environmental factor which seriously influences its growth and development. It is urgent to explore the thermotolerance mechanism of grapevine. Transcription factors HSFA2 and MBF1c were reported to be closely related to thermotolerance in plants such as grapevine. However, it is not clear how they control the molecular regulatory network and how they interact each other in plant. Therefore, they may be important breakthrough points for the study of grape thermotolerance. The thermotolerance difference among some grape species (or cultivars) is very large. The previous studies usually use a cultivar as research object in each experiment, moreover, experimental treatments were simple. The comparative studies between heat sensitive and tolerant grapevines were less. The present study will be conducted on one year grapevines of heat susceptible ‘Jingxiu’ (Vitis vinifera L.) and heat tolerant ‘Tangwei’ (Vitis davida Foex). Firstly, it will be confirmed if the thermotolerance difference between the two grapevines is related to the sequences of HSFA2 and MBF1c, their encoded proteins, and transcriptional levels under direct heat stress and heat stress after heat acclimation. Secondly, it will be explored if the thermotolerance difference is related to the target genes and networks regulated by HSFA2 or MBF1c and the interaction between HSFA2 and MBF1c. At last, the thermotolerance function of HSFA2 and MBF1c will validated through transgenic methods in grapevine. We hope to preliminarily elucidate the effect of HSFA2 and MBF1c on the thermotolerance difference among grapevines, and lay the foundation for clarifying the molecular regulation network of grape thermotolerance.

葡萄是具有重要经济价值的果树，高温是制约其生长发育的关键环境因素之一，迫切需要深入研究其抗热性机理。转录因子HSFA2和MBF1c与葡萄等植物抗热性关系密切，但尚不明确它们调控的分子网络及相互关系，可能是葡萄抗热性研究的重要突破点。不同葡萄种（品种）之间抗热性差异很大，但以前研究往往试材单一、处理简单，极缺乏不同种或品种的比较分析。项目以抗热性强的刺葡萄‘塘尾’和抗热性弱的欧亚种葡萄‘京秀’为试材，首先明确两种葡萄HSFA2（MBF1c）基因结构和编码蛋白的差异，以及它们在直接高温胁迫和高温锻炼后高温胁迫下转录水平的差异；其次探究两种葡萄HSFA2（MBF1c）所直接调控目标基因的差异，以及HSFA2和MBF1c是否存在上下游转录调控或蛋白互作关系；然后对HSFA2和MBF1c进行功能验证。揭示HSFA2与MBF1c对不同葡萄抗热性差异的影响机制，为阐明葡萄抗热性分子调控网络奠定基础。

葡萄是世界上重要的果树，极端高温天气频繁出现，严重影响了其产量及品质。以耐热性弱的欧亚种葡萄‘京秀’和耐热强的刺葡萄‘塘尾’为研究试材，研究热激转录因子HSFA2和辅转录因子MBF1c耐热性调控机制。结果表明：1. ‘京秀’等和‘塘尾’具有不同的HSFA2单倍型，分别命名为VvHSFA2和VdHSFA2。VvHSFA2和VdHSFA2的启动子活性受高温诱导，且VdHSFA2的启动子活性高于VvHSFA2。高温处理后，VdHSFA2的表达上调倍数明显高于VvHSFA2。VvHSFA2和VdHSFA2蛋白具有7个氨基酸的差异，都具有转录激活活性。VdHSFA2的转录激活活性高于VvHSFA2，主要与这两者转录激活域的氨基酸变异（Thr315→Ile315）有关。在葡萄‘41B’悬浮细胞中过表达VvHSFA2和VdHSFA2均提高了葡萄的耐热性，且VdHSFA2提高葡萄耐热性的能力高于VvHSFA2；利用CRISPR/Cas9介导的基因编辑技术获得的hsfa2突变细胞系耐热性显著降低。此外，通过分析ChIP-Seq和RNA-Seq数据，发现VdHSFA2调控的下游靶基因远多于VvHSFA2。VvHSFA2和VdHSFA2的这些差异可能是导致欧亚种葡萄与刺葡萄耐热性差异的重要原因。2. MBF1c是HSFA2调控的下游靶基因。‘京秀’和‘塘尾’MBF1c的启动子和蛋白序列是相同的。ChIP-qPCR、酵母单杂交及双荧光素酶报告试验的结果表明，‘塘尾’VdHSFA2和‘京秀’VvHSFA2都可以结合并激活MBF1c的表达，且‘塘尾’VdHSFA2对MBF1c的调控能力高于‘京秀’VvHSFA2。MBF1c的表达受高温诱导，在葡萄中定位于细胞核，在酵母体系中没有检测到其转录激活活性，而在拟南芥原生质体中发现具有转录激活活性。在葡萄‘41B’悬浮细胞中过表达MBF1c增强了葡萄的耐热性，而利用CRISPR/Cas9介导的基因编辑技术获得的mbf1c突变细胞系耐热性显著降低。综上所述，HSFA2、MBF1c和HSPs通过转录水平和蛋白水平上调参与葡萄的耐热性；HSFA2基因的变异是刺葡萄比欧亚种葡萄具有较高的耐热性的重要原因，这种变异可用于葡萄耐热性杂交育种的早期选择以及分子育种；MBF1c作为HSFA2的直接下游靶基因对葡萄耐热性也起着正调控作用。