苹果树腐烂病菌非核糖体多肽合成酶基因VmNRPS12的功能

Function of nonribosomal peptide synthetase gene VmNRPS12 of Valsa mali

摘要:

[目的] 非核糖体多肽合成酶（NRPS）在植物病原真菌与其寄主互作过程中发挥着重要作用，明确VmNRPS12基因在苹果树腐烂病菌致病过程中的功能，将为今后深入研究苹果树腐烂病菌NRPS作用机制提供理论依据。[方法] 基于苹果树腐烂病菌全基因组数据，得到VmNRPS12基因。运用qRT-PCR技术分析VmNRPS12在侵染初期的表达水平，利用Double-joint PCR和PEG介导的原生质体转化获得该基因抗潮霉素的突变体，对突变体进行PCR检测及Southern blot验证得到敲除突变体，进一步通过重新导入该基因全长片段获得互补突变体，最后对野生型、敲除突变体和互补突变体进行菌落、产孢及致病力观察，对检测数据用SPSS软件进行差异显著性分析。[结果] 定量分析显示该基因在侵染初期显著上调表达，且接种48 h后的表达量是对照的138.6倍。该基因的敲除突变体在营养生长及产孢方面与野生型菌株03-8相比无显著性差异，但致病力与野生型菌株03-8相比显著减弱，且互补突变体致病力近似恢复至野生型水平。[结论] VmNRPS12基因与苹果树腐烂病菌致病性相关。

Abstract:

[Objective] Nonribosomal peptide synthetase (NRPS) plays an important role in plant-pathogenic fungi interaction. In this study, we analyzed the role of VmNRPS12 during Valsa mali (V. mali) infection and pathogenecity, which may shed new insights into the pathogenesis of V. mali.[Methods] Based on the genome of V. mali, we obtained one NPRS, designated as VmNRPS12. The expression profile of VmNRPS12 was analyzed by qRT-PCR. Through Double-joint PCR and PEG-mediated protoplast transformation, VmNRPS12-knockout mutants were generated. PCR and Southern blot hybridization were applied to verify the positive mutant. Then genetic complementation was performed by transforming mutant protoplast with VmNRPS12 original gene. Finally, vegetative growth, conidiation and pathogenicity were examined.[Results] qRT-PCR analyses showed that VmNRPS12 was upregulated during the early infection stages of V. mali, and expressed remarkably at 48 hours post inoculation (138.6-fold). Compared with the wild type 03-8, VmNRPS12-knockout mutant showed no obvious change in vegetative growth and conidiation on PDA medium. Notably, the VmNRPS12-knockout mutant exhibited significantly reduced virulence on apple twigs. Furthermore, complementation of VmNRPS12 restored the pathogenecity of the VmNRPS12-knockout mutant approximately to the wild type 03-8.[Conclusion] VmNRPS12 contributes positively to the pathogenicity of V. mali.