苹果树腐烂病菌果胶裂解酶基因Vmpl4的致病功能研究

发布时间：2019-01-28 22:26

【摘要】：【目的】通过研究苹果树腐烂病菌果胶裂解酶(pectate lyase,PL)基因Vmpl4敲除突变体的致病力、对果胶的利用和基因敲除后家族内其他基因的表达水平变化,探明Vmpl4在致病过程中的作用。【方法】采用q RT-PCR技术检测Vmpl4在野生菌株03-8与寄主互作过程中的表达水平及Vmpl4敲除后PL家族内其他基因的表达水平,通过Doublejoint PCR构建基因敲除载体,并通过PEG介导原生质体遗传转化获取转化子、PCR及Southern blot验证基因敲除突变体。利用苹果叶片和枝条离体接种方法检测突变体致病力;接种至PDA及果胶培养基,观察突变体的营养生长和对果胶的利用情况。【结果】Vmpl4在病菌侵染过程中上调表达高达10.20倍;通过验证得到1个基因敲除突变体,其在叶片和枝条上的致病力均显著降低,在果胶培养基上生长速率也明显降低,Vmpl4敲除后家族内4个基因在病菌侵染过程中表达水平显著上调。【结论】果胶裂解酶基因Vmpl4通过降解果胶参与致病过程,PL家族内其他基因与Vmpl4在病菌致病性方面共同发挥作用。
[Abstract]:[objective] to study the pathogenicity of Vmpl4 knockout mutants of Pectin lyase (pectate lyase,PL) gene of apple tree rot pathogen, and to investigate the changes of Pectin utilization and the expression level of other genes in the family after gene knockout. [methods] Q RT-PCR technique was used to detect the expression level of Vmpl4 in the interaction between wild strain 03-8 and host, and the expression level of other genes in PL family after Vmpl4 knockout. The gene knockout vector was constructed by Doublejoint PCR, and the transformant was obtained by PEG mediated protoplast genetic transformation. The mutant was verified by PCR and Southern blot. The virulence of the mutant was detected by in vitro inoculation of apple leaves and branches. After inoculation with PDA and pectin medium, the nutrient growth and utilization of pectin were observed. [results] the expression of Vmpl4 was up to 10.20 times during infection. The pathogenicity of a gene knockout mutant was significantly decreased in leaves and branches, and the growth rate in pectin medium was also significantly decreased. The expression of four genes in the family after Vmpl4 knockout was significantly up-regulated during infection. [conclusion] Pectin lyase gene Vmpl4 is involved in the pathogenicity through the degradation of pectin. Other genes in the PL family and Vmpl4 play a role in pathogenicity.
【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】：国家自然科学基金(31171796和31471732) 国家公益性行业(农业)科研专项(201203034) 高等学校博士学科点基金(20120204110002)
【分类号】：S436.611.11

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