Isolation And Functional Analysis Of VmHMG7 Gene On Valsa Mali Var.Mali

Valsa mali var.mali is a widespread and harmful pathogen cause apple canker disease.In-depth study of the growth and development and pathogenic molecular mechanism can provide theoretical basis for scientific prevention and control of the disease.Isolate and clone the genes that regulate the growth and development of pathogens,and study the function of these genes can reveal the growth and pathogenesis of rot pathogen.In this study,the ATMT-mediated mutant FL550 were selected as the starting strains,which has a great different with the wild strains on colony morphology,growth and pathogenicity.By the amplification of flanking sequences,the HMG-box genes were isolated and cloned.Using bioinformatics methods,the HMG-box family of apple rot pathogen was identified from the whole genome database of Valsa mali var.mali,and the genetic family was analyzed by MEGA5 software.The structure and chromosome localization of gene family members were carried out by Map DRAW and GSDS.Using double-joint PCR technique to construct the knockout vectors,which have the Valsa mali var.mali HMG（VmHMG7）gene upstream and downstream fragments and the screening marker gene（Hygromycin phosphotransferase Gene,HPH）.The knockout vectors were transformed into the wild strains protoplast by PEG-CaCl₂.Then,through the screening of hygromycin,PCR amplification and Southern hybridization,the knockout mutants were obtain.Compared the morphology,growth,pathogenicity and phenotypic with the wild strain,the function of HMG-box gene were preliminary analysis.The results are as follows:1.Flanking sequence clone and sequence analysis showed that,the substitution site2336793 to 2341108 in chromosome 8（CM003105.1）was replaced by the T-DNA insertion of the ATMT vector in the mutant FL550.The total length of the replaced fragment is 4316bp,which includes the 5’termenal and its promoter of the VM1G₀7985（VmHMG7）,the partial promoter sequence of VM1G₀7986（KUI72125.1）,and the noncoding region sequence between this two genes.The VM1G₀7985,which is located in the chromosome 8（GenBank:CM003105.1）of Valsa mali var.mali,have a high homology of 99%with the HMG（High Mobility Group-box containing protein）gene of sunflower stem ulcer bacteria（Diaporthe helianthi）,so that named it as VmHMG7 gene.The DNA length is 2327bp,encoding 631amino acid hypothetical protein.VM1G₀7986 encodes a 1,3-beta-glucan synthase gene.This study focuses on the VmHMG7 gene and analyzed the HMG-box gene family.2.Whole genomes data analysis showed that Valsa mali var.mali included 8 HMG-box genes,inclued VmHMG1（1107bp）,VmHMG2（1509bp）,VmHMG4（897bp）,VmHMG3（300bp）,KUI27382.1（1752bp）,VmHMG6（1374bp）,VmHMG8（606bp）,VmHMG7（1893bp）.The chromosome 2 contains 4 HMG-box genes（VmHMG2,VmHMG3,VmHMG4,VmHMG5）.VmHMG1,VmHMG6,VmHMG7,VmHMG8 are in the chromosome 1,3,8,9,respectively.The 8 HMG-box genes are are listed to two subfamilies:HMGB-UBF（VmHMG1,VmHMG2,VmHMG4,VmHMG3）and MATA（VmHMG5,VmHMG6,VmHMG8）.3.The results of genomic analysis showed that the VmHMG7 gene contained three domains:Peptidases_S8_S53,Sprt-like（DNA metalloproteinase）and HMG-box.The VmHMG7 gene was similar to PaHMG7（accession number CDP29961.1）,which also have Sprt-like and HMG-box domains.The similarity is to 51%.The main effect of PaHMG7 is on the distribution of fruiting bodies and has no effect on colony morphology.It is assumed that VmHMG7 has a similar function.4.Screening and phenotypic analysis of VmHMG7 gene knockout strainsΔ1-5 andΔ2-22 revealed that the pathogenicity was significantly improved,knockout efficiency is2.1%.There are not different between the wild strains and the mutants on colony morphology,size and spore-producing.But the average growth rate of mutants decreased by 10.8%and19.8%respectively,and the pathogenicity increased by 48.5%and 56.2%,which indicated that the deletion of VmHMG7 gene inhibited the growth of Valsa mali var.mali,enhanced its pathogenicity,and had no obvious effect on other traits.