枯萎病菌侵染时香蕉异分支酸合酶基因的表达调控机制

Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense (Foc), is a devastating soil-borne fungal disease that is one of the most destructive plant diseases in the world. Banana production has been severely affected by Fusarium wilt. Salicylic acid is one of important signal molecules, inducing plant systemic acquired resistance to defense the infection of pathogens. In our previous study results suggest that one reason for banana susceptibility to Foc TR4 is SA biosynthesis was suppressed. Isochorismate synthase (MaICS) is one of the key gene in the isochorismate branch pathway to salicylic acid biosynthesis in banana (Musa acuminata L. AAA group, cv. Cavendish). However, the regulatory mechanism of MaICS gene expression in banana under Foc TR4 infection is not clear, which limits the further application of salicylic acid through activating its biosynthesis to increase the resistance to Foc TR4. In this study, we intend to expression the MaICS protein using Pichia yeast expression system, and to identify the function of MaICS in salicylic acid biosynthesis. And then, Cis-acting regulatory elements of MaICS gene will be explored by cloning MaICS gene promoter and 5′end deletion mutants. The yeast one-hybrid experiment will be performed to screen transcription factors directly regulating the expression of MaICS gene. And finally, we intend to induce the MaICS gene expression using hormones, environmental factors or elicitors in vitro to improve the resistance ability of banana to Foc TR4. This work will reveal the regulatory mechanisms of MaICS gene expression during salicylic acid biosynthesis under Foc TR4 infection, and will provide a theoretical basis for regulating the salicylic acid biosynthesis to induce resistant to Foc TR4 infection by biotechnology.

香蕉枯萎病是一种典型的土传病害，严重影响香蕉的种植。水杨酸是诱发植物系统获得抗性的重要信号分子。我们前期研究发现水杨酸的生物合成未激活是香蕉易感枯萎病的原因之一；香蕉异分支酸合酶（MaICS）基因是水杨酸合成途径的关键酶。但在枯萎病菌侵染时MaICS基因表达的调控机制不清楚，在生产中限制通过调控其表达提高香蕉抗枯萎病的应用。本项目拟采用毕赤酵母表达系统表达MaICS蛋白，鉴定枯萎病菌毒素对其酶活性的影响；通过克隆MaICS基因启动子结合5′端缺失突变发掘调控MaICS基因表达的响应元件，应用酵母单杂交方法分离调控该基因表达的转录因子；筛选能激活响应元件和转录因子的激素、环境因子或激发子激活MaICS基因的表达进而提高香蕉对枯萎病的抗性。本研究将揭示枯萎病菌侵染下MaICS基因在香蕉水杨酸生物合成中表达的调控机制，为通过体外调控技术调节香蕉水杨酸生物合成，提高香蕉抗枯萎病能力提供理论依据。

香蕉是重要的热带亚热带水果，是重要的经济作物。香蕉枯萎病是一种毁灭性土传真菌病害，严重威胁香蕉产业健康发展。水杨酸参与香蕉抗枯萎病过程。异分支酸合成酶（Isochorismate synthase, ICS）是水杨酸生物合成的关键酶之一，也是水杨酸生物合成的限速酶。ICS可将莽草酸途径中的分支酸变构为异分支酸，再经丙酮酸裂解酶的作用将其分解，形成水杨酸。在香蕉A基因组中，MaICS以单拷贝存在，因此MaICS基因是控制香蕉水杨酸生物合成的关键基因。本项研究采用RT-PCR的方法，克隆了ICS基因在巴西蕉中的全长序列，研究了ICS基因在的酶活和基因表达在抗病、感病香蕉品种接种Foc TR4中的表达趋势，发现MaICS基因参与香蕉抗枯萎病过程；利用RT-PCR技术分离MaICS基因启动子序列，分析顺式作用元件，采用酵母单杂交方法分离获得5个可能的调控因子。MaWRKY1可以与MaICS启动子互作进而可能负调控MaICS基因的表达。在大肠杆菌中表达MaARF1重组蛋白，经镍柱纯化后获得MaARF1重组蛋白，进行EMSA实验，结果表明MaICS基因启动子探针与MaARF1重组蛋白存在互作。研究结果为后续MaICS基因在香蕉抗枯萎病过程中的机制奠定基础。