苹果树腐烂病菌细胞色素P450基因Vmcyp5的功能

Characterization of cytochrome P450 gene Vmcyp5 in Valsa mali

摘要:

[目的]研究表明，细胞色素P450（CYP）在死体营养型真菌的毒素合成代谢中发挥重要作用，预测可能与病原菌致病相关。论文对苹果树腐烂病菌（Valsa mali）毒素合成基因簇中的1个上调表达的CYP基因Vmcyp5进行生物学功能研究，明确CYP基因对病原菌致病力影响，为细胞色素P450基因家族对苹果树腐烂病菌致病机理的进一步研究提供依据。[方法]通过Double-joint PCR和PEG介导的原生质体转化技术获得具有G418抗性的突变体，并对突变体进行PCR检测及Southern blotting验证得到单拷贝敲除突变体。将目的基因片段重新导入敲除突变体，筛选获得互补突变体。最终对野生型菌株及敲除突变体、互补突变体进行菌落、产孢及致病力观察，利用SPSS软件对数据进行差异显著性分析，并利用qRT-PCR技术分析突变体黑色素基因簇的表达水平。[结果]通过基因敲除技术获得1个Vmcyp5基因的敲除突变体。与野生型菌株相比，Vmcyp5基因的敲除突变体菌落呈白色，产孢量减少51.3%。qRT-PCR分析发现敲除突变体黑色素基因簇基因表达量降低。重要的是，敲除突变体致病力较野生型菌株降低24.5%。互补突变体菌落颜色、产孢及致病力近似恢复至野生型菌株水平。[结论]Vmcyp5基因与病原菌黑色素合成、子实体的产生和致病力相关。

Abstract:

[Objective] Cytochrome P450 (CYP) plays a significant role in mycotoxin metabolism of necrotrophic fungus and may be related to pathogenicity. The objective of the study was to reveal the function of CYP genes in infection process.[Methods] The knockout cassette was constructed using Double-joint PCR and mutants were obtained and confirmed by PEG-mediated transformation of protoplasts, PCR and Southern blotting analysis. Gene complemented mutants were constructed by gap repair technology. The PDA routine culture was selected to analyze the vegetative growth of mutants. In vitro inoculation to apple twigs was used to detect the pathogenicity. Salient differences were analyzed by SPSS and qRT-PCR was used to detect the expression of melanin gene cluster.[Results] Compared with the original strain 03-8, the colony color of deletion mutant changed from light yellow to white, and the amount of propagulum reduced by 51.3%. The relative expression level of Vmcyp5-knockout mutant was assayed by qRT-PCR, and mutant's melanin gene cluster was down-regulated. More importantly, the pathogenicity of deletion mutant was decreased by 24.5%. Complementation mutant was almost back to the level of the original strain 03-8 in colony color, propagulum and pathogenicity.[Conclusion] Vmcyp5 may be related to melanin biosynthesis and propagulum formation, and participates in pathogen process of Valsa mali.