论文部分内容阅读
【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Vm NRPS12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。
【Objective】 Non-ribosomal polypeptide synthetase (NRPS) plays an important role in the interaction between plant pathogenic fungi and their host. To clarify the function of Vm NRPS12 gene in the pathogenic process of apple tree rot will further study the effect of apple tree To provide a theoretical basis for the action mechanism of rot bacteria NRPS. 【Method】 VmNRPS12 gene was obtained based on whole genome data of apple tree rot. The expression level of VmNRPS12 in the early stage of infection was analyzed by qRT-PCR. Mutants of hygromycin resistant to hygromycin were obtained by Double-joint PCR and PEG-mediated protoplast transformation. The PCR products of the mutants were confirmed by PCR and Southern blot Mutants were knocked out and the complementary mutants were further obtained by reintroducing the full-length fragment of the gene. Finally, colony, sporulation and pathogenicity were observed on the wild type, knockout mutants and complementary mutants. The test data were analyzed by SPSS software Difference significance analysis. 【Result】 Quantitative analysis showed that the gene was significantly up-regulated in the early stage of infection, and the expression was 138.6-fold after 48 h of inoculation. The knock-out mutant of this gene showed no significant difference in vegetative growth and sporulation compared with the wild-type strain 03-8, but the pathogenicity was significantly weaker than that of the wild-type strain 03-8, and the mutation of the complementary mutant The approximate return to wild-type level of force. 【Conclusion】 The VmNRPS12 gene is associated with pathogenicity of apple tree rot.