Agrobacterium Tumefaciens-Mediated Transformation Of Valsa Malivar. Maliand Generation Of A Transformant Library

Valsa mali var.mali(Vmm),the causal agent of Valsa canker of apple tree,has been a serious threat to apple production in shaanxi province and even in whole China.Understanding pathogeniciy-related genes will provide better understanding of pathogenic mechanisms of Vmm,which may contribute to more effective management of Valsa canker.However,pathogeniciy-related genes have not been studied so far.Agrobacterium tumefaciens-mediated transformation(ATMT)was widely used as a tool to study the pathogenicity-related genes of fungi because of its simplicity and convenience.So,in order to explore the pathogenicity-related genes of Vmm,A.tumefaciens-mediated transformation was first applied on Vmm,A.tumefaciens EHA105 carrying pBIG2RHPH2-GFP-GUS plasmid was used to transform the conidia of Vmm.The transformation system was established and optimized and an ATMT transformant library was generated and evaluated.The transformation system and transformant library will lay a good technical and material basis for exploring the pathogencity-related genes of Vmm.The main results were shown as follows:1.Transformation system was established,and filter membrane used in transformation experiments and cultivation condition of A.tumefaciens was optimized.The results indicated(1)the filter membrane was not essential for successful transformation;(2)MM cultivation of A.tumefaciens can be replaced by LB cultivation;(3)AS(Acetosyringone)added in IM did not influence transformation efficiency;(4)IM cultivation of A.tumefaciens can be omitted.The simplest transformation system was LB-CM.2.Through optimizing the factors:co-cultivation time and OD660 of A.tumefaciens,the optimal transformation system was as follows:after the overnight LB cultivation,A.tumefaciens was adjusted to OD660=0.3,and then mixed with the conidia of Vmm(106/mL),the mixture was spread on CM for 2 d,then,CM was covered by PDA with appropriate antibiotics.The transformation efficiency was about 26 transformants/105 conidia.3.Since gfp(green fluorescent protein)expression could not be detected in randomly-selected transformants transformed by A.tumefaciens EHA105 carrying pBIG2RHPH2-GFP-GUS plasmid,A.tumefaciens SK1044 carrying pSK1044 plasmid was used to transform Vmm,and the results showed Vmm was labelled by gfp successfully.4.A library contained about 6000 transformants was generated,the library was evaluated.97.6%(234/249)randomly-selected transformants showed growth on PDA with 60 μg/mL hygromycin B after 5-time cultivation on PDA without any antibiotics.This indicated transformants were mitotically stable;PCR analysis of hph gene in 20 randomly-selected transformants indicated hph(hygromycin B phosphotransferase)was integrated into the genome of Vmm;Southern blot analyses of 18 randomly-selected pathogenicity transformants indicated all pathogenicity transformants showed one or more hph fragment signals at various positions of nucleotide length,and 27.8%(5/18)pathogenicity mutants were single-copy T-DNA insertion mutants.