乙烯响应因子ERF057和ERF080调控葡萄抗寒性的机理研究

发布时间：2018-10-31 16:47

【摘要】：葡萄(Vitis vinifera L.)是我国重要的果树作物之一,北方产区冬季低温干旱,而栽培的主要品种抗寒性较差,冬季需埋土防寒,大幅增加了生产成本和种植风险。研究表明,气体激素乙烯在植物生物及非生物胁迫应答反应中发挥重要作用,乙烯信号通路下游的ERF (ETHYLENE RESPONSE FACTOR)转录因子可以调控胁迫相关基因的表达,从而提高植物的抗逆性。本研究以山葡萄(V.amurensis)和‘玫瑰香’(V. vinifera cv. Muscat Hamburg)组培苗为材料,研究了低温胁迫与乙烯生物合成的关系,及外源乙烯前体1-氨基环丙烷羧酸(1-aminocyclopropane-1-carboxylicacid, ACC)和乙烯合成抑制剂氨基乙基乙烯基甘氨酸(aminoethoxyvinylglycine, AVG)对葡萄抗寒性的影响；同时分别在两种葡萄中克隆了ERF转录因子ERF057和ERF080,对其在不同胁迫条件下的表达模式进行分析,通过异位表达研究了VaERF057的功能并利用RNA-seq分析其可能调控的下游基因。取得的主要研究结果如下：1、低温胁迫下,对山葡萄和‘玫瑰香’的乙烯释放量、ACC含量和ACO活性进行测定,结果表明低温胁迫可以促进ACC的合成并激活ACO的活性,从而导致葡萄中内源乙烯的合成速率增加。通过测定相对电导率计算半致死温度来评价山葡萄和‘玫瑰香’经ACC和AVG处理后抗寒性的变化,结果表明外源添加ACC可以显著提高山葡萄和‘玫瑰香’的抗寒性,而添加AVG却增加了山葡萄和‘玫瑰香’对低温的敏感性,以上结果表明乙烯正调控葡萄对低温胁迫的耐受性。2、根据参考基因组序列,分别在山葡萄和‘玫瑰香’中克隆了两个ERF转录因子ERF057和ERF080。经过分析发现,ERF057和ERF080均含有1个高度保守的由59个氨基酸组成的AP2/ERF结构域。ERF057和ERF080分别属于第Ⅶ亚类和第Ⅸ亚类ERF转录因子,它们可能具有与各自同源蛋白相似的生物学功能,参与葡萄中生物与非生物胁迫应答。3、利用荧光定量RT-PCR对ERF057和ERF080在不同胁迫处理下的表达模式进行分析,结果表明,低温和外源添加ACC均能诱导山葡萄和‘玫瑰香’中ERF057和ERF080的上调表达,而添加AVG则完全抑制低温对ERF057和ERF080表达的诱导作用。由于低温可以诱导葡萄内源乙烯的加速合成,因此推论ERF057和ERF080在低温条件下的上调表达可能依赖于内源乙烯的生物合成。4、利用农杆菌介导法将VaERF057和VaERF080转入拟南芥,结果表明过表达VaERF057和VaERF080显著提高了拟南芥的抗冻性。转基因拟南芥植株中的MDA含量显著低于野生型和空载,而SOD.POD和CAT的活性显著高于野生型和空载,表明VaERF057和VaERF080可能通过降低植株膜脂过氧化的程度和提高其去除ROS的能力来提高转基因拟南芥的抗寒性。5、利用酵母单杂交技术证明了VaERF057具有与GCC-box和DRE元件结合的能力。转录组分析表明过表达VaERF057导致拟南芥89个基因表达显著上调,51个基因表达显著下调,这些基因包括低温信号通路关键基因以及乙烯信号途径下游基因。启动子分析表明有24个基因含有GCC-box,有54个基因至少含有1个DRE核心序列。由此表明VaERF057可能通过结合GCC-box或DRE元件,调控其下游基因的表达,来参与转基因植株对低温胁迫的应答。
[Abstract]:Vitis Verifera L. is one of the important fruit tree crops in China, and the winter low temperature drought in the northern region, while the main varieties of the cultivation are poor, the winter needs to be buried, and the production cost and planting risk are greatly increased. The research shows that the effect of gas-hormone ethylene plays an important role in the response of plant biology and abiotic stress, and ERF (ERF) transcription factor downstream of ethylene signaling pathway can regulate the expression of stress-related genes, thus improving the stress resistance of plants. In this study, Vitis viniferae (V. amurensis) and Flos Rosae Rugosae (V. vinifera cv. The effects of low temperature stress on the biosynthesis of ethylene biosynthesis, and the effects of exogenous ethylene precursor 1-aminocyclopropane-1-carboxyllic acid (ACC) and ethylene synthesis inhibitor, aminoethylvinylglycine (AVG) on grape yield were studied. ERF transcription factor ERRF057 and ERF080 were cloned into two grapes, and the expression patterns were analyzed under different stress conditions. The function of VaERRF057 was studied by ectopic expression and the possible downstream gene was analyzed by RNA-seq. The main results obtained were as follows: 1. Under low temperature stress, the grape and Flos Rosae Rugosae Ethylene release, ACC content and ACO activity were measured. The results showed that low temperature stress could promote the synthesis of ACC and activate the activity of ACO, which resulted in an increase in the synthesis rate of endogenous ethylene in grapes. evaluation of mountain grapes by measuring the relative conductivity and calculating the semi-lethal temperature Flos Rosae Rugosae The results of ACC and AVG treatment showed that the addition of ACC could significantly improve the quality of Vitis vinifera. Flos Rosae Rugosae The addition of AVG adds to the mountain grapes and Flos Rosae Rugosae the above results show that ethylene is controlling the tolerance of grapes to low temperature stress. Flos Rosae Rugosae Two ERF transcription factors ERRF057 and ERF080 were cloned. It was found that ERF057 and ERF080 each contain 1 highly conserved AP2/ ERF domain consisting of 59 amino acids. ERRF057 and ERF080, respectively, belong to the ERF transcription factors of the class VII and the second subclasses, which may have biological functions similar to their respective homologous proteins, participate in biological and non-biological stress responses in grapes, and analyze the expression patterns of ERRF057 and ERF080 under different stress treatments using fluorescence quantitative RT-PCR. The results showed that both low temperature and exogenous ACC could induce mountain grape and Flos Rosae Rugosae The up-regulated expression of ERRF057 and ERF080, while the addition of AVG, completely inhibited the induction of low temperature on ERRF057 and ERRF080 expression. Since low temperature can induce the accelerated synthesis of endogenous ethylene in grapes, it is deduced that the upregulation of ERRF057 and ERF080 under low temperature conditions may depend on the biosynthesis of endogenous ethylene. 4. VaERR057 and VaERRF080 are transferred to Arabidopsis using Agrobacterium-mediated method. The results showed that the expression of VaERRF057 and VaERF080 significantly improved the frost resistance of Arabidopsis thaliana. The content of MDA in transgenic Arabidopsis plants was significantly lower than that of wild type and no-load, while the activity of SOD, POD and CAT was significantly higher than that of wild-type and no-load, indicating that VaERRF057 and VaERRF080 could improve the tolerance of transgenic Arabidopsis by reducing the degree of lipid peroxidation of plant membrane and enhancing its ability to remove ROS. The ability of VaERR057 to bind to GCC-box and DRE elements was demonstrated by using yeast single hybridization technique. Transcriptional group analysis indicated that the expression of VaERR057 resulted in a significant increase in the expression of 89 genes in Arabidopsis, and the expression of 51 genes was down-regulated. These genes included the key genes of low-temperature signal pathway and the downstream gene of the ethylene signaling pathway. The promoter analysis indicated that 24 genes contained GCC-box, and 54 genes had at least one DRE core sequence. It is suggested that VaERRF057 can regulate the expression of downstream gene by binding GCC-box or DRE element to participate in the response of transgenic plant to low temperature stress.
【学位授予单位】：中国科学院研究生院(武汉植物园)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S663.1

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