苹果树腐烂病菌T-DNA插入突变体的筛选及Vmzfp3基因的功能分析

摘要

本研究采用接种离体枝条的方法,对150个苹果树腐烂病菌的T-DNA插入突变体库中的突变体进行了致病性的筛选,经过多次试验得到了3个致病力明显减弱的突变体,编号为X4379,X4368,X4468.基因组重测序结果显示,突变体X4379有2个T-DNA插入位点,分别是锌指蛋白3(Vmzfp3)和跨膜蛋白42 (Vmtme42).以苹果树腐烂病菌的全基因组序列为依托,设计引物,构建敲除载体以及互补表达载体.运用PEG的方法转化苹果树腐烂病菌,分别获得Vmzfp3和Vmtme42的敲除转化子和互补转化子.对Vmzfp3和Vmtme42基因敲除转化子、互补转化子以及野生型苹果树腐烂病菌菌丝形态和致病力进行分析.与野生型菌株03-8相比,Vmzfp3的敲除转化子的菌丝生长速率明显减慢,致病力也明显下降,其互补转化子使得这些功能缺陷均得以恢复.Vmtme42的敲除转化子无明显区别.由此证明基因Vmzfp3对病菌的菌丝生长,病菌的致病力具有调控作用.%This research identified three T-DNA mutants(X4379,X4368 and X4468)which displayed reduced pathogenicity from the ATMT mutant library by inoculating apple twigs.X4379 was chosen to do the following research.Sequences of the results showed that there are two T-DNA insertion sites,which are zinc finger protein with KRAB and SCAN domains 3 (Vmzfp3)and transmembrane protein 42 (Vmtme42).The primers were designed according to the genome sequence of Valsa mali to construct the targeted gene knockout vector and fusion expression vector.Finally,we got the targeted gene knockout transformants ΔVmzfp3,ΔVmtme42 and complementary transformants(ΔVmzfp3-c,ΔVmtme42-c) using PEG method.We evaluated the characteristics such as the mycelium morphology and the pathogenicity of transformants.The results displayed that the Vmzfp3 kncokout transformants had slower mycelia growth rate on PDA medium,and the pathogenicity also significantly weakened,but the complementary transformants had a great consistency with the wildtype strains 03-8.The Vmtme42 knockout transformants had no defects in mycelium morphology and pathogenicity.The results suggest that Vmzfp3 has regulatory functions to mycelium morphology and pathogenicity.It can conclude that the gene Vmzfp3 may affect mycelial growth of V.mali.