| Lactobacillus plantarum was one of the strongest Lactobacillus species of environmental adaptability,which was a model species for Lactobacillus research.The naturally plasmid-containing Lactobacillus plantarum was of great significance for its survival.Application and research on Lactobacillus plantarum were promoted by studying its natural plasmid.Lactobacillus plantarum belonged to gram-positive bacterium,which's cell wall was thicker and which was difficult to be broken,so the plasmid extraction rate was low.It seriously affected the research and application of plasmid from gram-positive bacteria.In terms of plasmids sizes,diversities and quantities,Lactobacillus plantarum was notable.Lactobacillus plantarum PC518 strain harbored multiple plasmids,but it was difficult to isolate and purity.This study optimized the extraction method of plasmid from Lactobacillus plantarum,increased the efficiency of plasmid extraction,and found a method for detecting multi-plasmid system in bacteria.The analysis of plasmids from Lactobacillus plantarum could enrich the database of natural plasmids and lay the foundation for systematic study of natural plasmids.Objective1.To improve a method for plasmid extraction and find a way to increase the extraction rate of plasmid from gram-positive bacteria.2.Detection of new plasmids from Lactobacillus plantarum PC518 strain.3.The new plasmid was annotated and sequenced.Materials and Methods1.Improvement a method for plasmid extraction: modifying the concentration,time and temperature of lysozyme treatment,the removal of lysozyme.It optimized a method for plasmid extraction from gram-positive bacteria.Plasmid DNA of Lactobacillus plantarum PC518,410,9L15,and JS193 and Weissella cibaria M2 were extracted using the gram-positive bacteria plasmid isolation kit(Solarbio,Beijing,China)and our modified method based on the Omega kit respectively.2.The plasmid from Lactobacillus plantarum PC518 was extracted by modified plasmid extraction method,and the new plasmid was detected using plasmid DNA library,inverse PCR and BLAST at NCBI website.3.DNA star,Mega X and QPCR were used to annotate the plasmid,analyze its sequence and copy number.Results1.The modified plasmid extraction method improved the lysozyme concentration to 100 mg/ml,shortened the treatment time to 10 min,and increased the washing step by 5 % glucose solution to remove lysozyme.The plasmid concentrations of the tested strains increased by 10.6,9.5,1.5,6 and 5.6 times respectively using the modified method for plasmid extraction,and the plasmid bands increased significantly.2.A new plasmid pLP60 was detected from Lactobacillus plantarum PC518.3.Plasmid pLP60 was classified as class A of theta replication.The copy number of which was measured as 5 copies per cell,and it contained eight ORFs,including a replication protein(RepB)and two mobile proteins.The ori contained an AT-rich region and a Rep-binding site.The former contained short tandem repeats(sTR),which repeated 4.1 times and the latter contained long tandem repeats(lTR),which repeated 4.4 times.The phylogenetic tree was constructed and the similarity rates of Rep proteins of 22 plasmids in the evolutionary tree with repB of pLP60 are greater than 45 %.Rep cluster analysis showed that the ori may not have sTR,but it must have lTR which was conservative.These plasmids were classified into six classes,according to the conservation of lTR.Conclusion1.The step of lysozyme removal is the key to improve the efficiency of plasmid extraction.2.The multi-plasmid system in bacteria was detected using plasmid DNA library,inverse PCR and BLAST at NCBI website.In the classification of class A plasmid pLP60 of theta replication,it was found that the traditional method by Rep classification was not applicable,and the classification by lTR of the ori was more effective. |
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