MADS-box转录因子Rlm调控拮抗酵母菌抗逆性和生防效力的分子机制

Antagonistic yeasts, naturally occurring on fruit surfaces, have been proven to control fruit diseases effectively and safely. However, during application it is inevitable for them to face a wide array of environmental stresses that impact their biocontrol performance. Studying the function of transcription factor has become a hot topic in the research field of plant pathology. The knock-out mutant of MADS-box transcription factor Rlm of the antagonistic yeast, Candida oleophila, was generated. The preliminary results indicated that the stress resistance of the mutant to high temperature and oxidative stress was significantly depressed, compared to wild type strain. Moreover, the biocontrol efficacy of the mutant against blue and gray mold in apple and kiwifruit also decreased. In order to unveil the regulation network of Rlm in stress resistance and biocontrol efficacy, this project plans to analyze the target genes regulated by Rlm and the Rlm-interacting proteins, respectively. Differentially-expressed genes were analyzed between the mutant and wild type strains through the proteome and transcriptome analysis. The techniques of yeast two-hybrid assay, chromatin immunoprecipitation assay (ChIP), electrophoretic mobility shift assay (EMSA) and bimolecular fluorescence complementation (BiFC), were employed to find out the target genes regulated by Rlm and the Rlm-interacting proteins. This study will provide the new theoretical basis for highly efficient development and utilization of antagonistic yeasts in the biocontrol of fruit diseases.

自然存在于水果表面的拮抗酵母菌被证实可安全有效地防治水果病害。然而，在施用过程中拮抗酵母菌会遇到各种逆境胁迫，从而影响其生防效果。研究具有重要功能的转录因子的调控机制正逐渐成为植物病理学领域中的热点。本实验初步研究发现拮抗酵母菌（Candida oleophila）MADS-box转录因子Rlm的功能缺失会造成拮抗菌对高温和氧化逆境的耐受性下降，并导致对苹果、猕猴桃果实青霉病和灰霉病的防治效果显著降低。本项目拟利用转录组和蛋白质组学的方法分析突变株中差异表达的基因，通过酵母双杂交、染色质免疫共沉淀、凝胶阻滞和双分子荧光互补等方法探寻Rlm调控的靶标基因和与之互作的蛋白，并进一步验证其功能。最终解析转录因子Rlm调控拮抗酵母菌C. oleophila抗逆性和生防效力的分子机制，进而为拮抗菌在水果病害生物防治中更为有效的开发利用提供理论基础。

近年来，基于天然拮抗菌的生物防治技术逐渐成为替代化学农药防治水果采后病害的有效措施。在诸多拮抗菌中，自然存在于水果表面的酵母菌由于不产生抗生素等特性而备受研究关注。本项目在解析了拮抗酵母Candida oleophila遗传图谱的基础上，成功获得转录因子Rlm的突变体菌株。通过抗逆性和生防效力的检测发现，转录因子Rlm突变体的对高温、氧化和高盐等逆境的耐受性，以及对苹果、猕猴桃等水果的采后真菌病害的防治效果，均显著低于野生型菌株。通过比较转录组分析Rlm突变体和野生型，挖掘Rlm的靶基因，以及相关生理生化技术鉴定互作蛋白。通过优化培养基中金属离子的配比，激活拮抗酵母Candida diversa转录因子Rlm，增强细胞壁强度，进而增强有效生物量以及对苹果青霉病的防效。该项目解析了转录因子Rlm对酵母菌的抗逆性和生防效力的调节机制，为拮抗菌的高效开发利用奠定理论基础。