果胶甲酯酶(PME)在香蕉抗枯萎病过程中的作用及机理研究

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the most destructive diseases in the production of banana (Musa spp.) in the world. Breeding of resistant cultivars and the study of associated mechanism are the major task for banana production. The plant cell walls form the first obstacle that a pathogen needs to overcome. To overcome this obstacle, pathogens secrete numerous cell wall degrading enzymes (CWDEs) to degrade the pectins in the plant cell wall. The degree of methylesterification (DM) of pectins and the distribution of individual pectin components with different DM have been correlated with changes in the susceptibility of plants to pathogens. Plant pectin methylesterase (PME) were involved with plant resistance to the pathogens throung afttecting the DM status of pectins. However, the machanism underlying is far from understanding. In the present study, the tissue-cultured plants and suspension cells fo two banana cultivars, highly resistant and susceptible to this disease respectively, are selected as plant materials to study the resistant mechanism. The following parameters will be investigated: (1) Analysis of the activity and transcription level of PME, the DM of total pectins; (2)The changes in spatial distribution and abundance of individual cell wall pectins and PME in roots before and after pathogen infection using immuno-labelling techniques with 11 types of monoclonal antibodies recognizing different epitopes of pectins at different DM and PME of banana; (3) Changes in the satus of pectin DM (including celluar distribution and abundance of individual cell wall pectins at different DM), PME activity after immuno-modulation of PME, and test of resistance of the treated plants to this disease after pathogen infection. The goal of this study is to gain a better and more complete understanding of the role of PME in the methyl-esterification status of pectins and resistance to Foc in banana-Foc interaction, which will be benefit for the breeding of banana cultivars resistant to Fusarium wilt.

香蕉枯萎病是当今世界香蕉生产的最严重威胁。抗枯萎病育种及抗性机理研究是香蕉科研工作的首要任务。病原菌要成功侵染寄主，首先必须降解植物细胞壁果胶，细胞壁果胶甲酯化状态与抗病性密切相关。植物果胶甲酯酶（PME）通过影响果胶甲酯化状态进而影响植物抗病性。然而，其作用机理仍不清楚。本项目拟分别以高抗香蕉枯萎病的'粤优抗1号'和高感品种'巴西蕉'的组培苗和悬浮细胞为试材，（1）比较分析寄主与病原菌互作过程中，香蕉PME活性与转录水平及果胶甲酯化程度变化；（2）利用PME及11种识别不同甲酯化程度果胶成份的单克隆抗体，采用免疫标记技术对接种病原菌前、后，细胞壁中相应抗原成份在香蕉抗、感品种中的时空分布及表达量差异进行研究；（3）通过免疫调控等方式调控香蕉中PME的表达量，研究调控后香蕉抗病性的变化，以揭示PME在香蕉-枯萎病镰刀菌互作过程中的抗病功能及作用机理，为抗枯萎病育种提供理论参考。

香蕉枯萎病是香蕉生产上的最严重病害，研究香蕉抗病机理是香蕉科研工作的首要任务。病原菌要成功侵染寄主，首先必须降解植物细胞壁果胶，细胞壁果胶甲酯化状态与抗病性密切相关。植物果胶甲酯酶（PME）通过影响果胶甲酯化状态进而影响植物抗病性。然而，其作用机理仍不清楚。本项目拟分别以高抗香蕉枯萎病的‘粤优抗1号’和高感品种‘巴西蕉’的组培苗为试材进行下述研究：（1）比较分析寄主与病原菌互作过程中，香蕉PME活性与转录水平及果胶甲酯化程度变化；（2）利用PME及11种识别不同甲酯化程度果胶成份的单克隆抗体，采用免疫标记技术对接种病原菌前、后，细胞壁中相应抗原成份在香蕉抗、感品种中的时空分布及表达量差异进行研究；（3）通过免疫调控等方式调控香蕉中PME的表达量，研究调控后香蕉抗病性的变化。得到的主要研究结果如下：（１）抗病品种的起始果胶酶活性高于感病品种的而果胶甲酯化程度则正好相反，伤根后抗病品种的PME活性基本保持稳定，相反感病品种的则呈上升趋势；无论是抗病品种还是感病品种，病原菌侵染均导致其PME活性不同程度的下降：荧光定量PCR及免疫荧光标记技术所获得的结果与酶活性结果高度一致。表明后香蕉对伤根的不同反应与其抗病性密切相关，即受伤后能保持较低PME活性及较高DM的品种抗性较强。DGE结果表明在抗、感品种接菌前后共13个PME成员差异表达。（２）与抗病品种相比，感病品种的根组织细胞虽然在未切根对照及处理的的早期比抗病品种含有较多的高甲酯化程度果胶成份，但在处理后期却表现出相反的结果。与抗病品种相比，感病品种的根组织细胞壁中无论在未切根对照及处理的各个时期均含有较多的低甲酯化程度果胶成份。（３）采用PME抗体对香蕉抗、感品种进行根部PME表达量调控后，抗病品种PME的表达量表现下降而感病品种的表现上升，感病品种经调控后发病率下降，而抗病品种的发病率没有发生明显变化。本项目所获得的结果可以帮助揭示PME在香蕉-枯萎病镰刀菌互作过程中的抗病功能及作用机理，从而为抗枯萎病育种提供理论参考。