中文摘要：

【目的】研究pH信号通路（Pal）在重寄生真菌盾壳霉与寄主核盘菌互作过程中的作用。【方法】从盾壳霉全基因组信息中分析获得了6个Pal相关基因CmpalA、CmpalB、CmpalC、CmpalF、CmpalH和CmpalI的全编码序列和氨基酸序列,通过PEG介导的原生质转化技术获得了CmpalA、CmpalB、CmpalC、CmpalF和CmpalH等5个基因的敲除突变体,分析这些敲除突变体与野生型在菌落培养性状、重寄生能力、降解草酸能力、产生抗真菌物质能力等方面的差异。【结果】与野生型相比,在pH 6–8的条件下,5个Pal相关基因敲除突变体的菌丝生长受到显著抑制,这说明缺失Pal相关基因使盾壳霉对高pH值环境更加敏感。菌核重寄生试验发现5个Pal相关基因敲除突变体的重寄生能力均显著低于野生型。qRT-PCR试验结果表明,敲除Pal相关基因之后导致重寄生相关酶基因Cmch1、Cmg1和Cmsp1的表达量显著降低,而且pH信号通路下游的CmpacC基因的表达量也显著降低。Pal相关基因敲除突变体在pH 6条件下对草酸盐的降解能力显著高于野生型,同时这5个突变体在pH 8条件下产生抗真菌物质能力也显著高于野生型。【结论】pH信号通路相关基因的缺失影响盾壳霉对环境pH的响应。pH信号通路在盾壳霉与核盘菌互作中发挥重要作用,不仅影响盾壳霉的重寄生作用,而且还影响盾壳霉的草酸降解作用和抗真菌作用。

英文摘要：

[Objectives] To identify pH-signalling pathway （Pal）-related genes in mycoparasitic fungus Coniothyrium minitans and to understand the role of these genes in interaction between C. minitans and its host Sclerotinia sclerotiorum. [Methods]Six Pal-related homologues were obtained from the whole genome of C. minitans and designated as CmpalA, CmpalB, CmpalC, CmpalF, CmpalH and CmpalL PEG-mediated protoplast transformation was used to create the deletion mutants of Pal-related genes. Five Pal-related genes were knocked out individually and the mutants designated as ACmpalA-33, ACmpalB-13, ACmpalC-5, ACmpalF-50 and ACmpalH-26. The biological characteristics, including colony morphology, mycoparasitism, oxalate degradation and antifungal activity,were compared between knock-out mutants and the wild-type strain. [Results] Compared to the wild type strain, five Pal-related genes-deletion mutants showed significantly reduced mycelia growth between pH 6 and 8. These results indicated that the disruption of these Pal-related genes increases sensitivity to neutral or alkaline pH. The sclerotia-infection assay showed that the parasitic activities of the five Pal-related genes-deletion mutants were dramatically reduced, qRT-PCR results showed that these Pal-related genes-deletion mutants suppressed expression levels of three mycoparasitism-associated genes Cmchl, Cmgl and Cmspl. Meanwhile, expression of CmpaeC, the pH signaling pathway downstream gene, was also reduced in the Pal-related genes-deletion mutants. The oxalate degradation of the five Pal-related genes-deletion mutants at pH 6 were increased under pH 8, and the antifungal activity of those mutants were also increased at pH 8 comparison with the wild type. [Conclusion] Disruption of the Pal-related genes resulted in impaired C. minitans responses to ambient pH. The pH-signalling pathway （Pal） plays an important role in interaction between C. minitans and S. sclerotiorum, including mycoparasitism, oxalate degradation and antifungal activity in C. minitans aga