苹果轮纹病菌(Botryosphaeria dothidea)果胶裂解酶基因Bdpl1的致病功能分析

 由葡萄座腔菌(Botryosphaeria dothidea)引起的苹果轮纹病是影响我国苹果安全生产的重大病害之一。因此,本研究基于受苹果轮纹菌侵染的苹果组织中果胶裂解酶基因Bdpl1表达上调这一现象,通过Split-marker PCR技术构建Bdpl1基因敲除载体,并通过PEG介导的原生质体转化获得转化子,经常规PCR和qRT-PCR对所获得的转化子进行筛选,成功获得1个Bdpl1基因缺失阳性突变子。该转化子在PDA上的培养基性状与野生型没有明显差异,但在果胶培养基上菌落直径明显小于野生型。其胞外果胶酶活相比野生型明显下降,但在离体“早富”苹果枝条上的致病力并没有明显的下降。通过qRT-PCR技术发现在基因Bdpl1敲除后,其家族内有3个基因在病菌侵染过程中相比野生型明显上调表达(>3倍)。这些现象表明果胶裂解酶基因Bdpl1与病原菌的营养生长过程关系不大,但其参与对寄主果胶类物质的降解。Bdpl1基因对轮纹病菌致病力的影响较小,有可能是Bdpl1基因敲除后,该基因家族内其他基因的上调表达补偿了Bdpl1基因的功能。

Pathogenic function analysis of pectin lyase gene Bdpl1 of Botryosphaeria dothidea in apple tree

Apple ring rot caused by Botryosphaeria dothidea is one of the devastating diseases in China, which has become a major threat to apple production. In preliminary experiments, we found the expression of pectin lyase gene (Bdpl1) was obviously up-regulated in diseased tissues than that in fungal mycelia. Therefore, the Bdpl1 was considered as a virulence gene involved in the pathogenic processes. In order to analyze its pathogenic function, the knockout vector of Bdpl1 was constructed by the split-marker method and the transformants were obtained through PEG-mediated protoplast transformation. One mutant, △Bdpl1-3, was confirmed with four pairs of primers. Compared with the wild-type strain, the △Bdpl1-3 morphology had no significant differences on PDA medium, whereas the colony diameter on pectin medium was much smaller than the wild-type one. The extracellular pectinase activity of the △Bdpl1-3 isolate was lower than the wild type. Nevertheless, pathogenicity of the △Bdpl1-3 isolate on excised one-year-old ‘Fuji’ apple twigs had no significant differences with the wild-type strain.Relative expression levels of several other pectin lyase (PL) family genes in the △Bdpl1-3 and wild-type strains were detected by qRT-PCR. The results showed that the expression of three other PL family genes were up-regulated (over three folds than the wild-type strain). These data indicated that the Bdpl1 had no obviously effects on fungal growth and pathogenicity, but it had significant acceleration to degrade pectin. In the △Bdpl1-3 strain, other PL family genes might compensate the functions of Bdpl1 by up-regulated expression.