苹果树腐烂病菌分隔蛋白基因VmImp2的功能研究

旨在研究苹果树腐烂病菌(Valsamali) VmImp2基因的功能,以期揭示腐烂病菌的生长发育及致病机理。利用生物信息学软件对 VmImp2进行蛋白序列及进化分析;利用Double-joint PCR和PEG介导原生质转化的方法,获得腐烂病菌 VmImp2的缺失突变体和回补菌株;利用十字交叉法以及伤口接种法,研究 VmImp2对病菌营养生长,繁殖体产生以及致病力的影响。生物信息学分析显示, VmImp2编码1 487个氨基酸,编码蛋白属于PCH(Pombe Cdc15 homology)家族,含有典型的FCH(Fes/CIP homology, F-BAR)和SH3(Src Homology 3)结构域,与梨树腐烂病菌(V.pyri)的Imp2蛋白亲缘最近。对基因进行敲除后,共获得5个缺失突变体菌株。表型分析显示,缺失突变体的营养生长受到明显影响,表现为菌丝更加稀疏,生长速率明显减缓。同时,突变体的繁殖体产生能力以及对枝条的侵染能力基本丧失。5个突变体的表型一致。将基因回补之后,共获得3个回补菌株,突变体表型缺陷基本恢复到野生型水平。说明 VmImp2基因参与苹果树腐烂病菌的营养生长、繁殖体形成以及致病过程。

Functional Study of Septation Protein Gene VmImp2 in Valsa mali

The septation protein Imp2 (Increased Maximal Permissive Tempera-ture for Pim1) plays an important role in fungal cell membrane trafficking and cytokinesis.Exploring the function of VmImp2 in Valsa mali is of great significance to further clarify the mechanism of vegetative growth and the propagules production,the pathogenicity of V.mali.bioinformatics analysis of the VmImp2 protein was performed using bioinformatics softwares.The double-joint PCR and PEG-mediated protoplast transformation technique were used to construct the gene deletion and complement mutants.The cross measurement and inoculation on apple twigs by wounding were used to explore the function of VmImp2 on vegetative growth.The propagules production and pathogenicity of V.mali.Bioinformatics analysis indicated that VmImp2 belonged to the PCH (Pombe Cdc15 homology) family and contained two typical domains of FCH (Fes/CIP homology,F-BAR) and SH3(Src Homology3). VmImp2 encoded 1487 amino acids,and it had the highest similarity with the Imp2 protein from V.piri.In total,five gene deletion mutants were obtained.Phenotypic analysis revealed that the vegetative growth of the deletion mutant strains changed significantly,the mycelium became sparser,and the growth rate significantly slowed down,meanwhile,the ability of propagules production and pathogenicity were lost,the phenotype of five mutants were identical.Further, VmImp2 was transformed back into the mutant strain,and three complementary strains were obtained.There is no significant difference between the complement strains and the wild type 03-8 strains based on the phenotypic analysis. VmImp2 is involved in regulating the vegetative growth,propagules production,and the pathogenicity of V.mali.