大豆ERF家族基因的克隆及抗逆机制的研究

Soybean is an important grain and economical crop which is commonly grown worldwidely. It was difficult to effectively improve stress resistance of soybean using traditional breeding methods. Elucidating the mechanism of soybean resistance and cloning the relative genes are important for improving stress resistance of soybean and other crops to acquired new varieties with excellent resistance. Studies have shown that soybean contains a large number of ERF transcription factors and the ERF transcription factor genes in plants can effectively improve the stress resistance of the transgenic plants. In view of this, a great quantity of new ERF transcription factors will be cloned from soybean; the resistance identification of transgenic Arabidopsis will be performed to get effectively resistant ERF transcription factor genes; and the regulatory mechanism, transcriptional regulation activities and interacting proteins of these ERF genes will be clarified; the adversity induced promoters of these ERF genes will also be obtained. This study will provide the theoretical basis for the further application of the soybean ERF genes, and enrich plant gene resources of resistance, lay the foundation for the cultivation of stress-resistant varieties of soybean or other crops.

大豆是世界范围内普遍种植的重要粮食和经济作物。采用传统的育种方法往往难以有效地提高大豆的抗逆性。运用现代分子生物学技术，阐明大豆抗逆机制，克隆相关基因并利用这些基因来改良大豆乃至其他农作物的抗逆性以获得抗性优良的新品种具有重要的意义。已有研究表明: 大豆中含有大量的ERF转录因子，且ERF转录因子基因能有效提高转基因植物的抗逆性。鉴于此，本研究从大豆基因组中批量克隆新的ERF转录因子基因，通过对转基因拟南芥的抗逆性鉴定，获得有效的抗性ERF转录因子基因，研究其调控机制，明确其转录调节活性及其互作蛋白等，并获得的具有逆境诱导活性功能的启动子。此研究为大豆ERF基因的进一步应用提供理论基础，并丰富植物抗逆基因资源，为大豆或其他作物抗逆品种的培育奠定基础。

ERF（Ethylene Response Factor）转录因子广泛存在于植物中并且参与植物对生物及非生物胁迫的应答。大豆中含有大量ERF转录因子，但仅有少数进行了功能鉴定。本研究利用已知的ERF基因保守序列搜索NCBI和大豆基因组数据库，获取15个功能未知的大豆ERF基因序列。利用实时荧光定量PCR对15个ERF基因在逆境处理下的表达情况进行检测，其中GmERF9和GmERF10在逆境处理下的表达量明显升高。凝胶阻滞分析表明GmERF9与GCC-box顺式作用元件特异结合。酵母杂交实验、GUS组织化学染色及GUS荧光定量分析表明GmERF9具有转录激活能力。将GmERF9基因转化烟草，转基因烟草中5个抗逆相关基因（PR1、PR2、Osmotin、SAR8.2和NtERF5）的表达量升高。非生物胁迫下转基因烟草生理指标检测结果表明GmERF9提高了转基因烟草的抗旱性和抗寒性。克隆GmERF9的启动子序列（GmERF9P）并对GmERF9P的逆境诱导启动活性进行鉴定。GUS组织化学染色及实时荧光定量PCR结果表明GmERF9P具有低温诱导启动活性。基于以上实验，本研究获得一个新的抗旱和抗寒的大豆ERF转录因子基因和一个新的低温诱导型启动子，为大豆ERF基因的进一步应用提供理论基础，并丰富植物抗逆基因资源，为大豆或其他作物抗逆品种的培育奠定基础。