| Polychlorinated biphenyls(PCBs)with the properties of oxidation resistance,acid alkali resistance,heat resistance,etc.are widely used in industrial production,but PCBs have harmful effects on human body and carcinogenicity,due to its banned production.Bioremediation of PCBs is considered to have a promising approach,however,the higher concentration of biphenyls and PCBs negatively affects microorganisms growth,cell division,and toxic compounds accumulation in the metabolic process.Rhodococcus sp.R04 can effectively degrade biphenyl and PCBs.Previous studies of this group found that R04 with the concentration of biphenyl and PCBs increasing,the phenomenon of filamentous was obvious,the septum formation of abnormal,transcriptome sequencing also indicated that the expression yield of cell division related protein FtsZ(filamentous temperature-sensitive gene Z)was decreased.In order to study the function of FtsZ,fusion plasmid containing ftsZ gene and pET-21a vector was constructed by us,then the plasmid was transformed into E.coli BL21 cell for expression.The results showed that the morphology of the cell with empty vector pET-21a have no change,before and after IPTG induction,which is the same as that of the cell that expresses FtsZ before IPTG induction.Moreover,a large number of filamentous cells were presence with the increase of IPTG concentration.According to statistical analysis towards cell length in different IPTG concentration and induction time,we found that the longer of incubation time,the longer of filamentous cell length,after growth five hours,the length of filamentous have reached 46.2μm.This reflect that FtsZ overexpression can inhibit E.coli cell division activity,which causes cell division interference,cell cannot form cell division site,and thus the final filamentous cell was formed.In prokaryotic organism,FtsZ protein is an essential protein for cell division.In order to reveal the role of FtsZ in bacteria cell division,the location mode of FtsZ in vivo was studied in this project.The genes of ftsZ and gfp were inserted into pkl8mobsacB to construct combination vector containing upstream ftsZ gene,downstream ftsZ gene and gfp gene,and then,green fluorescence labeled cells were screened by flow sorter,incubation in biphenyl,fluorencence was significant.We found that the fluorescence surrounded and pointed to the cell membrane.This phenomenon not only studys metabolic reactions of biphenyl is avails,but also provides reference for Z ring position and FtsZ division mechanism.Chromosome walking technology was used to study the recombination type of R04 mutant strain,and the result showed that gfp gene were successfully combined onto the genome of R04.To study the biological characteristics of the mutant strain,the biomasses of wild and mutant strain were determined by spectrophotometry.Comparing with wild strain,the mutant showed longer time for reaching logarithmic phase and slower growth rate,it illustrates that gfp affects the growth and metabolism of wild R04. |
