Complete Genome Analysis Of Mycoplasma Synoviae Isolates From Sichuan And Its Functional Genes Mining

Mycoplasma Synoviae(MS)can infect chickens of all ages,often resulting in synovitis,swollen joints and impaired movement.MS infection has spread worldwide now,MS infection is equally serious in various regions of China,causing severe economic losses to the poultry industry.In this study,whole-genome sequencing and analysis of 7 MS isolated from Sichuan were performed,primers were designed for the conserved region of MS virulence gene Vlh A,and Insulated isothermal RT-PCR(ii PCR)was established for rapid detection of MS.Furthermore,based on the cloning and expression of the MS virulence gene Glyc-eraldehyde-3-Phosphate Dehydrogenase(GAPDH),whether the expressed protein is related to MS adhesion to host cells was studied.Through the above research,the following results are obtained:1.Complete genome sequencing and comparative analysis of 7 mycoplasma synovial isolates from Sichuan were completed,and the genetic diversity of the isolates from Sichuan was confirmedIn order to understand the genetic diversity of Mycoplasma synoviae(MS),seven MS strains isolated from Sichuan were sequenced and analyzed by bioinformatics.Illumina Hi Seq platform and PE library were combined for whole-genome sequencing,and the whole genome was annotated by several databases.The complete genomes of 7 MS isolates were compared with those of 8 MS strains included in NCBI by MEGAX software.The results showed that the gene numbers,species and virulence factors of 4 MS strains MS251、MS254、MS221、MS231 isolated from the same chicken farm were the same or had little difference,while the gene number and virulence factor numbers of MS isolates MS12 H,MS1G and MSK1 isolated from other three different chicken farms were quite different.Comparing the 7 MS Sichuan isolates with the 8 MS strains published by NCBI,we found that the Sichuan MS isolates were significantly different from Brazilian MS53,American WVU-1853,Australian 86079/7NS and vaccine strain MS-H,however,most similar to Henan HN01,China.The genetic diversity of MS isolates in Sichuan was abundant in different fields and in the same field.At the same time,MS MLST typing showed that 7 MS isolates from Sichuan contained three ST types: ST104,ST34 and ST172.These results confirmed the genomic characteristics of MS sichuan strain,It provides a theoretical basis for the research of pathogenicity of MS isolates,the development of MS subunit vaccine and the establishment of specific detection technology.2.A fast ii PCR detection method based on MS Vlh A conservative region is established successfullyIn recent years,rapid diagnosis of MS has become more and more critical due to the spread of MS infection.For rapid and on-site detection of MS infection,a MS ii PCR detection method was established in this study.Based on the whole genome sequencing results of 7 MS isolates from Sichuan province and the whole genome analysis results of other MS isolates from NCBI,specific probes and primers were designed for the conservative region of Vlh A,and the reaction system and conditions were optimized.An ii PCR method was established to evaluate the specificity,sensitivity and stability of the method.The results showed that this method could only detect MS but not Mycoplasma gallinum,Avian Reovirus,chicken Escherichia coli,Salmonella enteriditis,Proteus mirabilis and Staphylococcus aureus,showing good specificity.The detection limit was 25.3 copies/μL of plasmid standard,indicating good sensitivity.And it has good repeatability.Twenty clinical samples of chickens with joint enlargement were collected from three farms.Compare the consistency between the ii PCR established in this study and the MS ii PCR method reported in the literature.The results showed that 16 samples were positive and 4 were negative,and the coincidence rate was 100%.Combined with DNA extraction kit,this method can be used to detect MS infection quickly.3.The involvement of MS GAPDH in adhesion to host cells was confirmedIn order to study whether MS GAPDH protein is involved in the adhesion of MS to host cells,the bioinformatics analysis of GAPDH gene of 7 MS Sichuan isolates was carried out.The complete sequence of GAPDH was obtained by designing primers and point mutation amplification and ligated into the expression vector p ET-32a+.The recombinant GAPDH protein(r GAPDH)was expressed in prokaryotic.Rabbit anti-r GAPDH high immune serum was prepared by immunizing rabbits with 500 μg/m L concentration of purified r GAPDH,and its Agar amplification titer was 25.1×10~6 CCU MS bacteria solution and MS bacteria solution were used to dilute rabbit anti-r GAPDH hyperimmune serum according to 1: 200,1:400 and 1:800 times to carry out adhesion and adhesion inhibition test.The results showed that the protein encoded by GAPDH gene had no transmembrane region and signal peptide and was easy to be expressed in prokaryotic cells.33.23% of the protein structure was irregular crimping.The Agar diffusion titers of serum antibodies at 14 days and 19 days after the second immunization of r GAPDH were 24 and 25,respectively.Adhesion and adhesion inhibition tests showed that MS could adhere to the host cell chicken fibroblast line(DF-1),while rabbit anti-r GAPDH high immune serum could significantly inhibit the adhesion of MS to DF-1.The results of this study are helpful to further understand the function of MS adhering to host cells.In conclusion,the whole genome sequencing analysis and MLST typing of 7 MS isolates from Sichuan confirmed the rich genetic diversity of MS isolates from Sichuan.Furthermore,the database analysis of virulence genes showed that the Vlh A gene had a high copy number in the MS genome,which could be used to establish detection methods based on its conserved region.Therefore,this study established ii PCR technology based on MS Vlh A gene for specific,sensitive and rapid detection of MS.GAPDH gene is also an important virulence gene,and it is involved in MS adhesion to host cells.This study confirmed that proteins encoded by GAPDH as a vital virulence gene of MS,participate in MS adhesion to host cells.It provides experimental data support for further study on the pathogenesis and prevention and control of MS.