苹果树腐烂病菌根皮苷水解酶基因Vmlph1的功能

摘要

为明确根皮苷降解酶在苹果树腐烂病菌侵染过程中的作用,从苹果树腐烂病菌基因组序列中鉴定出候选根皮苷降解酶基因Vmlph1,利用Double-joint PCR和PEG介导的原生质体转化技术构建Vmlph1敲除突变体,观察突变体营养生长状况、产孢情况以及致病力的变化;利用HPLC方法检测基因敲除对根皮苷降解速率的影响.结果发现,经PCR及Southern blot验证后获得Vmlph1基因的敲除突变体;与野生型菌株相比,突变体菌落颜色变白,生长速率和产孢能力显著降低;接种到叶片和枝条上,突变体致病力显著降低;突变体根皮苷降解能力与野生型没有显著差异;在根皮苷诱导下,野生型菌株黑色素合成调控转录因子Cmr1基因的表达量显著上调,但突变体Cmr1基因的上调倍数显著低于野生型菌株.表明,根皮苷降解酶基因Vmlph1与苹果树腐烂病菌营养生长、致病力、分生孢子的产生及黑色素合成有关.%To find out the importance of plorindzin degrading enzyme when apple trees were infected by valsa mail,the candidate plorindzin degrading enzyme gene Vmlph1 mutant was generated with double-joint PCR and PEG mediated protoplast transformation,the candidate mutant was later confirmed by PCR and southern blot.The results showed that compared to wild type,the virulence of plorindzin hydrolase gene deletion mutant reduced significantly both on leaves and twigs,and colony became white colour with slower growth rate and less conidiation.Furthermore,the HPLC results demonstrated that there was no difference in degradation ability of plorindzin between mutant and wild type,however,under the induction of phloridzin,theCmr1,a transcription factor regulating melanin synthesis,was significantly up-regulated expression in wild-type strain than deletion mutant.In conclusion,the phlorizin hydrolase gene Vmlph1 may have relationship with vegetative growth,participate in pathogenic process,conidiation and melanin biosynthesis of V.mali.