苹果树腐烂病菌多聚半乳糖醛酸酶基因Vmpg7和Vmpg8的功能

Function of Polygalacturonase Genes Vmpg7 and Vmpg8 of Valsa mali

【目的】前期转录组分析表明苹果树腐烂病菌(Valsa mali)两个多聚半乳糖醛酸酶(polygalacturonase,PG)基因Vmpg7和Vmpg8在病菌侵染定殖过程中显著上调表达。论文旨在分析Vmpg7和Vmpg8在病菌营养生长、致病力、果胶酶活性、对果胶的利用等方面的作用及基因双敲除对同家族内其他基因表达水平的影响,明确Vmpg7和Vmpg8在病菌致病过程中的生物学功能,为进一步解析苹果树腐烂病菌致病分子机理提供基础。【方法】通过q RT-PCR检测基因在病菌侵染过程中的表达水平及基因双敲除后PG家族内其他PG基因的表达量;利用double-joint PCR构建基因敲除载体并通过PEG介导的原生质体转化技术进行基因敲除,经4对引物PCR检测及Southern blot验证获得基因单敲除和双敲除突变体,并利用gap-repair技术对基因进行回复;利用PDA培养基常规培养法观察突变体营养生长状况;通过离体接种富士苹果叶片及枝条的方法检测基因缺失对病菌致病力的影响;通过3,5-二硝基水杨酸(DNS)比色法检测各突变体胞外果胶酶活性;利用Czapek培养基培养法分析突变体对果胶的利用情况。【结果】q RT-PCR分析表明,Vmpg7和Vmpg8在病菌侵染3 d后分别上调表达27.73和8.19倍。通过基因敲除技术,分别获得3个Vmpg7基因单敲除突变体、1个Vmpg8基因单敲除突变体和3个Vmpg7/Vmpg8基因双敲除突变体,并获得了Vmpg7和Vmpg8单敲除突变体的回复突变体。将突变体接种到PDA培养基上,菌落形态和生长速率均没有发生变化;接种到苹果叶片和枝条上,Vmpg7单敲除突变体及Vmpg7/Vmpg8双敲除突变体在叶片和枝条上的致病力显著降低,而Vmpg8单敲除突变体在叶片上的致病力显著降低;进一步对果胶酶活性及对果胶的利用分析,发现Vmpg8单敲除突变体及Vmpg7/Vmpg8双敲除突变体胞外果胶酶活性均显著降低,且所有突变体在果胶培养基上生长均明显减慢。此外,基因回复后突变体的致病力、果胶酶活性及在果胶上的生长速率均回复到野生型菌株03-8水平。重要的是,Vmpg7/Vmpg8双敲除后影响了PG家族中其他PG基因的表达水平,3个基因显著上调表达。【结论】多聚半乳糖醛酸酶基因Vmpg7和Vmpg8可能与同家族内其他基因协同作用,通过调节果胶酶活性参与苹果树腐烂病菌致病过程。

【 Objective 】 Transcriptome analysis showed that Vmpg7 and Vmpg8 are two significantly up-regulated polygalacturonase genes of Valsa mali during the pathogen infection progress. The objective of the study is to explore the role of these two genes in pathogenicity and lay the foundation for further pathogenesis of V. mali by analyzing the vegetative growth, pathogenicity, pectinase activity, the utilization of pectin, and the effect of gene knock-out of Vmpg7 and Vmpg8 to the expression levels of the other PG genes in PG family.【Method】q RT-PCR was used to detected the expression levels of Vmpg7 and Vmpg8 during the pathogen infection progress and the expression levels of other PG genes in PG family when Vmpg7 and Vmpg8 were deleted. The single and double deletion mutants were constructed and confirmed using double-joint PCR, PEG-mediated protoplast transformation technique, PCR with four pairs of primers and Southern blot, and the gene complemented mutants were constructed by gap repair technology. The PDA routine culture was selected to analyze the vegetative growth of mutants. In vitro inoculation to apple leaves and twigs was used to detect the pathogenicity. The 3,5-dinitrosalicylic acid（DNS） colorimetric method was used to detect the activity of extracellular pectinase, and the Czapek culture medium was selected to observe the utilization of pectin.【Result】The expression levels of Vmpg7 and Vmpg8 were assayed by q RT-PCR, and results showed that the two genes were up-regulated 27.73 and 8.19 folds at 3 days after infection, respectively. With the help of the gene knockout technique, three deletion mutants of Vmpg7, one deletion mutant of Vmpg8, three deletion mutants of Vmpg7/Vmpg8, together with the complemented mutants of Vmpg7 and Vmpg8 were constructed, respectively. When the mutants were cultured on PDA medium, the colony morphology and growth rate showed no significant change. When they were inoculated to apple leaves and twigs, the pathogenicity of the Vmpg7 deletion mutant and the Vmpg7/Vmpg8 double deletion mutant showed significant reduction, and the pathogenicity of the Vmpg8 deletion mutant was only reduced on apple leaves. Further analysis of pectinase activity and the utilization of pectin showed that the pectinase activities of the Vmpg8 deletion mutant and the Vmpg7/Vmpg8 double deletion mutant were significantly reduced, and all the mutants had a slow growth rate on pectin medium. Interestingly, the pathogenicity, pectinase activity, and the growth rate on pectin medium were back to the level of wild-type 03-8 when the genes were complemented back to the distinct mutants, respectively. More importantly, the expression levels of the other PG genes in PG family were obviously affected when Vmpg7 and Vmpg8 were knockout, especially three genes that were significantly up-regulated.【Conclusion】Vmpg7 and Vmpg8 participate in pathogenic process of V. mali by adjusting the pectinase activity, which may synergized with the other PG genes in the same family.