Isolation,Sequencing And Bioinformatics Analysis Of Yersinia Pestis’ 12mDa Plasmids Of Yunnan

Objective:To isolate, sequence and bioinformatics analyze the specific plasmids of Yersinia pestis in Yunnan for exploring the function and biological significance of the atypical plasmids in Yunnan.Methods:1. The Yersinia pestis strains were revived in selective medium, and then passaged in blood LB medium twice.2. Plasmids were extracted from strains by Kits and according to the method of Kado&Liu respectively and were separated by agarose gel electrophoresis.3. We used a clean blade to cut separated plasmid strip from the gel under UV lamp to obtain the plasmid DNA from gel by a series of experiments. Comparing most of the methods reported in the domestic literatures about gel extraction, we selected a total of 8 kinds of schemes, including using Gel Extract Kit, freezing and squeezing,using plastic fiber, using dialysis membrane, trenching, electric elution, low melting agarose abstract and saturated phenol extraction.4. The qualified plasmid DNA was sequenced.5. The sequence was given bioinformatics analysis including Genomic Component, Gene Prediction and Annotation, and Comparative Genomic analysis.Results:1. The strains growed well in Yersinia selective medium and then in blood LB medium twice.2. The plasmids were extracted both by the Kit and the Kado&Liu method. We obtained complete plasmid map in which strips less than 65 m Da were high concentration but strips more than 65 m Da were not clear by the Kit. We obtained plasmid map without 36 m Da plasmid in which strips were high concentration and clear by the Kado&Liu method. After overall consideration, we abstracted plasmids from Strain 2179 by the Kit while Strain 341, 611, 1247 by the method of Kado&Liu.3. Among the 8 kinds of gel extract schemes, we could obtain plasmid DNA only by the trenching method, Kit method and electric elution. After identification, only the12 m Da plasmid DNA was qualified for sequencing and the remaining samples were not qualified.4. 12 m Da plasmid sample was sheared by ultrasound, and the gene library less than 800 bp was successfully established, and then 10 Mb data was produced through the Hiseq2000 Illumina sequencing platform. Base on the sequencing data, a circular plasmid was assembled with 16465 bp genome size, 47.9% GC content. The sequence was one scaffold, one contig and belonged to the complete graph.5. The genome contained 25 ORFs accounting for 82.98% of the total length of the genome, with the total length of 13662 bp and the average length of 546 bp. There were 13 ORFs(accounting for 52%) that can be functional annotated in all the predicted ORFs base on Swiss-Prot and COG database. According to the functional annotation results, 12 m Da plasmid was found to contain pla and pst gene that encoded plasminogen activation factor and pesticin respectively in Yersinia pestis conventional plasmid p PCP. Transposable elements including the insertion sequence IS100 and arsenic resistant transposon were annotated.6. We synthesized the results of BLASTn and structural variation obtained by MUMmer software, and determined that the 12 m Da plasmid was Yersinia pestis conventional plasmid p PCP acquired insert sequences and transposon. There was a segment of gene difference that encoded and inactivated derivatives L of transposase between 12 m Da plasmid and Yersinia pestis JAVA9 plasmid p PCP.7. Phylogenetic trees and the consensus trees were produced by MEGA6 software. The results of gene collinearity homologous comparison between 12 m Da plasmid and other plasmids p PCP were done by MUMmer software.Conclusion:We have finished sequencing and bioinformatics analysis of the unique 12 m Da plasmid of Yersinia pestis in Yunnan. The 12 m Da plasmid is identified as a Yersinia pestis plasmid p PCP obtained the insertion sequence and the gene which expresses arsenic resistance.