Draft Genome Sequencing Of Pseudomonas Fluorescens AHK-1 And Functional Analysis Of The Candidate FliG Gene Associated With Pathogenicity

Pseudomonas fluorescens is a gram-negative bacterium widely found in the soil.It has multiple properties such as biological control,plant health promotion and causing plant diseases.This study is genome sequencing and analysis for the pathogenic P.fluorescens AHK-1 related to kiwi canker disease,to explore the virulence factors associated with plant diseases by comparing genomics and virulence factor prediction.Using the method of homologous recombination,we constructed the mutant strain and complementary strain and the biological function of fliG gene in the pathogenic P.fluorescens AHK-1 strain was analyzed.The research progress is summarized as follow:1.Draft genome sequencing of P.fluorescens AHK-1 In this study,draft genome sequence of P.fluorescens AHK-1 was obtained by the Illumina HiSeq 2500 sequencing platform.A draft genome sequence of 7,035,786 bp consisting of 50 scaffolds was obtained(GeneBank accession number:QRBA00000000),GC content is 60.88%,the largest scaffold is 626.42 kb,and the N50 size is 331.26 kb.A total of 6327 protein coding genes,4 rRNA and 58 tRNAs are predicted.2.Comparative genomic analysis of P.fluorescens AHK-1 The draft genome sequence of AHK-1 was compared with the P.fluorescens GcM5-1A and P.fluorescens SBW25,which were selected from the GeneBank database and the whole genome sequence was published.The results of orthologous gene analysis indicated that 3 strains shared 3998 core-genomes and 946 genes unique to strain AHK-1.In this study,19 strains of P.fluorescens including AHK-1 strain were selected for multilocus(acsA,aroE,guaA,gyrB,mutL,ppsA,pyrC,recA,rpoB)sequence analysis,19 The strain was divided into two sub-branches,in which the strain AHK-1 was closely clustered into a cluster with 16 strains of P.fluorescens.3.Functional analysis of the candidate fliG gene associated with pathogenicity The fliG gene deletion mutant strain ?fliG and complementary strain C-fliG of P.fluorescens AHK-1 were constructed by homologous recombination method.The results of biological test analysis showed that compared with wild-type strain,the motility and clustering abilities of mutant ?fliG were significantly reduced,decreased extracellular polysaccharides and biofilm formation ability,while complementary strains C-fliG can basically restore to the level of wild-type strain.After inoculation of the shoots of potted kiwifruit,the pathogenicity of ?fliG to kiwifruit was weakened,while AHK-1 and C-fliG showed strong pathogenicity to the inoculated shoots.This indicates that the flagellar-related gene fliG regulates the motility,extracellular polysaccharide and biofilm formation ability and the pathogenicity to kiwifruit of P.fluorescens AHK-1...