苹果树腐烂病菌非核糖体多肽合成酶基因VmNRPS12的功能

发布时间：2018-06-18 08:27

本文选题：Valsa + mali　；参考：《微生物学报》2016年08期

【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。
[Abstract]:[objective] Non-ribosomal polypeptide synthase (NRPS) plays an important role in the interaction between plant pathogenic fungi and its host, and the function of Vm NRPS12 gene in the pathogenicity of apple tree rot pathogen is clarified. It will provide a theoretical basis for further study on the mechanism of NRPS in apple tree rot pathogen. [methods] VmNRPS12 gene was obtained based on the whole genome data of apple tree rot pathogen. The expression level of VmNRPS12 in the early stage of infection was analyzed by qRT-PCR. The mutant resistant to hygromycin was obtained by Double-joint PCR and PEG-mediated protoplast transformation. The mutant was detected by PCR and Southern blot validation. Further, the complementary mutants were obtained by reintroducing the full-length fragment of the gene, and the colony, sporulation and pathogenicity of wild type, knockout mutants and complementary mutants were observed. [results] quantitative analysis showed that the expression of the gene was significantly up-regulated at the early stage of infection, and the expression level was 138.6 times higher than that of the control 48 h after inoculation. There was no significant difference between the knockout mutant and wild strain 03-8 in vegetative growth and sporulation, but the pathogenicity of the mutant was significantly weaker than that of wild strain 03-8. And the pathogenicity of the complementary mutants was approximately restored to the wild-type level. [conclusion] VmNRPS12 gene is related to the pathogenicity of apple tree rot bacteria.
【作者单位】：西北农林科技大学植物保护学院旱区作物逆境生物学国家重点实验室;
【基金】：公益性行业(农业)科研专项(201203034) 国家自然科学基金(31171796) Ⅲ计划(B07049)~~
【分类号】：S436.611.11

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