Sterile Culturing And Whole Genome Sequencing Of Microcystis Aeruginosa XW01

Microcystis blooms have caused huge environmental disaster.In order to study the molecular biology of Microcystis,it is a critical step to obtain the complete genome sequence of Microcystis,which requires to sterilize Microcystis strains with large number of bacteria.Microcystis aeruginosa XW01 was taken as experimental material,physical purification methods including centrifugation washing method,dilution method,ultrasonic shock washing method and UV irradiation sterilization method,chemical purification(antibiotic treatment)were used to purify XW01.5 species antibiotic including ampicillin(Amp),kanamycin(KA),gentamycin sulfate(Gm),tetracycline(TE),neomycin(N)were chose as experimental drugs.To study the effect of different concentrations of different antibiotics on XW01 growth,OD650 of XW01 and Chi a content were measured.The main epiphytic bacteria were isolated and cultured.Their sensitivity to 5 kinds of antibiotics and the best processing condition of antibiotics were studied.To choose the best irradiation time to attain the sterilizing XW01.ultraviolet(UV)radiation was used on algae and bacteria.In this study the mixed antibiotics or(and)UV irradiation was able to eliminate plenty epiphytic bacteria from Microcystis colonies.The results showed that the centrifugal washing method with dilute method,ultrasonic washing method sterilizing effect respectively high 68%,22%,so this method as auxiliary measures purified microcystis.The bacteria were highly sensitive to Neomycin and Gentamycin.Then liquid culture of M.aeruginosa XW01 was with 5?g/mL N and 5?g/mL Gm for 24 h.At last by washing away antibiotics with fresh BG11 medium,an axenic XW01 culture was obtained.On the other study,the artificial UV at 1.25 ?W/cm2(30min)radiation can remove 99.99%of epiphytic bacteria.The way of using mixed antibiotics combined with 10min UV irration was more efficient to obtain axenic strain.Comparing with the mixed antibiotics treatment,the XW01 cells were easy to survive treated with the combing method.Genomic DNA was extracted from XW01,based on the assemble result of XW01,the genome size was 5,086,097 bp,GC content was 42.34%,the number of scaffold was 49,and the number of contig was 115.From the genome analysis results of XW01,we found that the genome contained 5,187 genes,the total length of genes was 4,187,682 bp,which makes up 82.34%of genome,the number of tandem repeat sequence was 1,303.the total length of tandem repeat sequence was 136,813 bp,which makes up 2.69%of genome,the number of minisatellite DNA was 413,the number of microsatellite DNA was 546,the number of tRNA was 48,the number of rRNA was 9.755 Mb data was produced for XW01.Then the results of linear synteny analysis of XW01 was compared with the reference M.aeruginosa NIE843 and M.aeruginosa PCC7806 at the nucleic acid and animo acid levels.The results showed that the three nucleic acid sequence had a lot of similarities and the amino acid sequence XW01 was more similar arrangement with PCC7806.The results ofMicrocystis purification methods provide a new way for later sterile study.The whole genome data of XW01 lay an important foundation for subsequent molecular work.