Pathogenicity Of Vibrio Harveyi And Cloning And Deleting Of T6SS Relate Gene

Vibrio Harveyi, as a main pathogen bacterium in mariculture, can infect many kinds of fish, shrimp, crab and cause a lot of death, which is highly value in aquaculture at present. Type VI secretion system(T6SS) as a new secretory system,which was found in gram negative bacteria in the past few years, which a lot of research prove that is a transmembrane protein secretion system associated with bacterial virulence. T6 SS, composed of a variety of protein complexes, transported effector proteins to the host cells when bacteria and host cell contact and have toxic effects on the host after. T6 SS was found in the majority of gram negative bacteria such as V.cholerae, Pseudomonas aeruginosa, V. alginolyticus, V. anguillarum, V. nguillarum, Rhizobium, Escherichia Coli, etc. In this paper, we study the drug resistance and pathogenicity of V. harveyi, vgr G gene and rbs B gene were cloned and analyzed to display the pathogenesis of T6 SS in V. harveyi. The pathogenesis mechanism was explored of build the transporters vgr G gene mutations in V. harveyi. The main results were showed as follows:1.128 strains were identified isolated from maricultured fish in the South China Sea. Phylogenetic trees were constructed using Neighbor-Jonining of series gene rct B-tox R, ultimately determine the 90 strains of V. harveyi. The drug resistance temporal and spatial distribute were tested by Kirby-bauer diffusion method in total of 90 V. harveyi strains. Phylogenetic of drug resistance was analyzed by squared Euclidean distance using bacteriostatic rings diameter of 12 kinds of antibiotics. The diversity of the drug resistance spectrum of 90 strains was discussed in this report. Results showed that 90 V. harveyi strains formed 26 antibiogram types and antibiogram abundance was 28.9%. 35 strains, occupied 38.9% of total strains, had 21 multi-drug antibiogram types(resistant to ≥ 3 antibiotics). The antibiogram types showed significant different of 90 V. harveyi strains in years and regions, which were 10, 18 and 8 in 2012, 2013 and 2014 years respectively, while 17, 13 and 2 kinds included in Hainan, Guangdong and Guangxi province correspondently. Phylogenetic and cluster analysis indicated that 90 strains separated to groupⅠ, group Ⅱ and others cluster. There were 11 antibiogram types with the abundance 16.9% in groupⅠ, 12 antibiogram types with 57.1% in group Ⅱ, while 4 kinds with 100% in others cluster. Subsequently groupⅠ and group Ⅱ could be divided into 5 subgroups of i~v. The results indicate that V. harveyi strains display complex antibiogram types in Hainan, Guangdong and Guangxi Province, and the resistance spectrum of V. harveyi not only change but also expand as time goes on.2.173 strains of V. harveyi infected Barchydanio rerio isolated from years 2007-2014 results showed that the mortality 100% of 45 strains, accounting to 26% of the total isolates; and 0% mortality of number 18 accounted for 10.4% of the total. The first two days is fast death of injected Barchydanio rerio, results indicate that V.harveyi is the rapid lethal pathogenic bacteria. Two pathogenic bacteria(X12XC30 and X13SZ03) was isolated from Pearl gentian in Guangdong and Hainan province. All the two isolates were identified as V. harveyi by biochemical tests and 16 S r DNA, rct B, tox R sequence analysis. Strains X12XC30 and X13SZ03 had significantly similar biochemical and physiology characteristics with V.harveyi and can naturally clustered together with the branch of V. harveyi. The strains,LD50 respectively 6.58×106 CFU·g-1 and 9.83×105 CFU.g-1,infected fishes by intraperitoneal injection. The results showed that virulence, V. harveyi, was confirmed by pathogenicity assays to pearl gentian, and endanger aquaculture industry.3.Vgr G gene and rbs B gene of T6 SS were amplified by PCR from V.harveyi genomic DNA, get a 930 bp ORF and 801 bp ORF, encoding 310 amino acide and 267 amino acide, respectively. Bioninformatics software Signal P 3.0 analysis reveal that vgr G protein of T6 SS transport protein containing a 1-39 aa signal peptide belongs to protein domain of “COG3501”. Rbs B, effect protein of T6 SS, was found the signal peptie area in 1-43 aa, and had “COG1879” protein structural domain.4.Upstream and downstream primers of vgr G gene were design by the http://ne builder.neb.com/ system contain overlap the same with plasmid primers p WS7848.V-UP, V-DOWN and p SW7848 fragments were got by PCR amplify. Recombina nt plasmid p SW7848-?vgr G was constructed by the isothermal recombination met hod and the culture of bacterial culture after two times transformation, then conj ugation with V. harveyi 170 EGH. Use of TCBS + 5 ug/m L Cm + 0.2% Glucose to screening the p SW7848-?vgr G recombinant plasmid of V. harveyi, Finally,th e plasmid p SW7848 carries the ccd B gene coding for a toxin, under the control of the arabinose inducible promoter p BAD. The ara C gene codes for the p BAD repressor. LBS+0.2% arabinose induced promoter p BAD control ccd B expressi on toxicity to kill plasmid and PCR amplify to obtain the final vgr G gene mutant strain of V.harveyi. The mutant mortality rate was significantly lower than the wi ld strains of V.harveyi infected Barchydanio rerio, respectively 70% and 30%. Th e results Suggest that vgr G gene of T6 SS play an important role in the process of V.harveyi pathogenic.