中文摘要：

【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。

英文摘要：

[Objective] The Apple Valsa canker, caused by Ascomycete Valsa mali, seriously affects the tree vigor and yield of apple in China. Previous studies inferred that pectinase might play an important role during the pathogenic process of V.mali. Pectate lyase （PL） is a class of pectinase. It cleaves uronic acid into an unsaturated hexenuronic acid and a new reducing end via a β-elimination mechanism. Transcriptome analysis showed that pectate lyase gene Vmpl4 was significantly up-regulated during infection of V.mali. The objective of this study is to verify the pathogenesis-related function of the pectate lyase gene Vmpl4 in V. mall, the utilization of pectin of Vmpl4 deletion mutant and the changes of relative expression levels of other genes in PL family when Vmpl4 was deleted. [ Methods ] One year old twigs of Ma- lus domestica borkh. ＇Fuji＇ and PDB （Potato Dextrose Broth） medium were inoculated with the wild-type strain 03-8. The lesion bark and the shaking cultured mycelium were collected three days post inoculation as the samples during the infection process and the control. Total RNA of the samples was extracted using the Nucleic acid extraction kit. The relative expression level of Fmpl4 during infection process was detected by quantitive real time PCR （qRT-PCR） with the glucose- 6- phosphate-dehydrogenase （G6PDH） gene as the inner reference gene. Genomic DNA of 03-8 was extracted by CTAB （Hexadecyl trimethyl ammonium Bromide）method. The upstream and downstream fragments of Vmpl4 were amplified by PCR using the DNA of 03-8 as template. Hygromycin B phosphotransferase gene （hph） was amplified from plasmid pHIG2RHPH2-GFP-GUS. Then Double-joint PCR was used to generate the gene deletion cassette by connecting the three fragments. The transformants were obtained through PEG-mediated proto- plast transformation technique, which led to the gene deletion cassette into the protoplasts of 03-8. PCR with four pairs of primers was used to verify the transformants by amplifying the target gen