Analysis Of Whole Genome And Pathogenic Related Gene Function Of Pectobacterium Carotovorum Subsp.brasiliense

Pectobacterium carotovorum is an important pathogen in plants,which has a wide range of hosts and has been found all over the world.This pathogen could cause severe economic damages in agriculture.With the development and popularization of DNA sequencing technology,the development of genomics has been accelerated.This technology is an effective method for studying genetic development and pathogenic mechanisms of bacteria by providing valuable information on genome content.To date,there is little report on the genomic information and pathogenic regulation mechanism of Pectobacterium carotovorum subsp.brasiliense.In this study,the whole genome of Pcb BZA12 strain was sequenced by DNA sequencing technology,and analysis of pathogenicity-related gene characteristics of pathogens from the genomic level by bioinformatics.Screen out the pathogenicity-related candidate effector gene,and explore its function.The main results are as follows.1.In this study,bacterial soft rot samples of cucumber and eggplant collected from different regions in Liaoning Province were isolated and cultured.Observation of colony morphology and identification of physiological,biochemical and molecular biology of bacteria.The result showed that the pathogenic bacteria isolated from cucumber and eggplant were Pectobacterium carotovorum subsp.brasiliense and Pectobacterium carotovorum subsp.carotovorum,respectively.All isolated bacteria were were no significant difference in colony morphology.Physiological and biochemical analysis showed that there were differences in utilization of alpha-methyl glucoside,D-sorbitol,acid production from maltose and sucrose reduction ability between the two pathogens.Molecular biological analysis showed that the strain isolated on cucumber can produce a 320 bp fragment under the amplification of Pcb specific primers,while the isolated strain on eggplant could not,but can produce a550 bp fragment under the amplification of Pcc specific primers.PCR-RFLP primer amplification and 16 Sr DNA phylogenetic tree construction all can distinguish between two pathogens.2.In this study,We present the complete genome sequence of Pcb BZA12 strain based on the combination of Hiseq 2500 Illumina and Pacbio RSII DNA sequencing technology,which Analyzed three aspects of Genomic component,gene function annotation and comparative genome.The result showed that Pcb BZA12 strain carries a single 4924809 bp chromosome with 51.97% GC content and comprises 4508 predicted protein-coding genes.A total of 192 tandem repeats,139 minisatellite sequences and 10 microsatellite sequences were predicted,and comprises 76 t RNAs,22 r RNAs and 26 s RNAs.The results of pathogenic gene analysis showed that there were 54 cell wall degrading enzyme-related genes,215 bacterial Ⅰ^Ⅵ secretory system-related genes and 16 toxin-related genes were predicted.Comparative genomic analysis showed that the number of gene families shared by three subspecies of P.stipitis was 3107,and the number of unique gene families of Pcb BZA12,Pco BC S7 and Pcc PC1 were 36,22 and 11,respectively.There are 3,859 consensus genes and 649 unique genes in the Pcb BZA12 strain.Pectobacterium phylogenetic tree analysis showed that the Pcb BZA12 strain and the other two Pcb strains had the closest genetic distance,and the genetic relationship with other strains of the same genus was relatively distant.3.We utilized three bacterial type Ⅲ effector protein sequence prediction software to predict effectors of Pcb BZA12,and we predicted five putative effector gene sequences based on functional annotation information and transiently expressed candidate effectors in N.benthamiana to characterize their sub-cellular location and their effects on host phenotype.Finally bioinformatics analysis of these genes.The results showed that only the candidate effector gene of PCB₂483 can induce cell death,and localized in the cell nucleus.Bioinformatics analysis showed that the gene PCB₂483 is 98 percent similar to the known gene of Pcb SX309 and Pcc PCC21 strains.This gene contains 305 amino acids and the largest proportion are basic amino acids.We found only one protein that may interact with it.The gene is a hydrophilic and high lipid protein with a large proportion of alpha helices and random coils in the secondary structure.4.We constructed a deletion mutant strain of PCB₂483 gene by using the T-linearized suicide vector p LP12 and the up-and-down homologous arm fusion gene of the target gene,and constructed a functional complement strain of the mutant strain.Together with the wild strains,comparative analysis of pathogenicity,allergic and some biological characteristics of three strains.The results showed that the motility and biofilm formation of the mutant strains were different from those of the wild-type and the complement strains.There were no significant differences in bacterial sedimentation,extracellular enzyme hydrolysis,hypersensitive response and pathogenicity.