Screening Of Imperfect Development Mutant Strains And Electrical Mutant Strains Of Dictyostelium Discoideum

The migration of cell is movement which is produced under the stimulus of migration signal. The symmetrical shape of cell transforms to asymmetrical, and cell arise leading edge and rear. These changes maintain the cell polarity and provide the premise for the efficiency of migration movement during the migration process. Cell migration process is divided into four steps: protruding of leading edge, adherence of pseudopodia on matrix substrate, cell migration and dragging the rear forward. In the numerous forms of cell migration, chemotaxis migration and electrotaxis migration are important ways in the processes of life. Chemotaxis migration refers to cells migrate directionally along the chemical concentration gradient by the chemotactic factor. Directional cell migration guided by small directed electric fields is known as electrotaxis/galvanotaxis. A large number of studies have shown that cell chemotaxis migration and electrotaxis migration exist widely in regeneration, tissue repair, inflammatory response, tumor metastasis and embryonic development, and they effect these processes of life.Dictyostelium discoideum was chosen as the research object.The restriction enzyme-mediated integration mutagenesis(REMI) was used to structure mutant library of Dictyostelium discoideum. The wild type of Dictyostelium discoideum and REMI mutant strains were used to development experiment and external electric field. The purpose was to screening REMI mutant library out mutant strains of developmental defect and mutant strains of electrotaxis. Through the screening of development experiment and electrotaxis experiment, we hope to explore the mechanism of development of Dictyostelium discoideum, cell chemotaxis migration and electrotaxis migration, and to promote the research progress in this field. The experiment contents and results are as follow:①Structuring the REMI mutant library of Dictyostelium discoideumThe restriction enzyme-mediated integration mutagenesis(REMI) was used to structure mutant library of Dictyostelium discoideum. First of all, the circular plasmid pBRS-1 was extracted which contain the resistance gene of BlasticidinS, and then restriction enzymes BamHI was used to enzyme cut pBRS-1 circular plasmid to linear DNA fragments with GATC sticky end.The second, the linear DNA fragments which has contain the resistance gene of BlasticidinS, wild type of Dictyostelium discoideum AX2 cell and restriction enzymes DpnII which cut the genome of Dictyostelium discoideum to linear DNA fragments with GATC sticky end were mixed to electroporation. The voltage is 0.85 kV, capacitance 0.25μF, interval of 5 seconds, a total of 2 times to electroporation. The purpose of this step is restriction enzymes DpnII cut randomly the genome of Dictyostelium discoideum with GATC sticky end, and the linear DNA fragments which had the same sticky end as BamHI integrate on genome Dictyostelium discoideum. The BlasticidinS was used to drug screening after electroporation, and the survivals were selected with Klebsiella aerogenes(KA) to bacterial culture and the plaques were picked. We had the second drug screening after bacterial culture, and its purpose was to eliminate false positive. If the Dictyostelium discoideum cell survived after twice drug screening, we determined the cells were mutant strain of Dictyostelium discoideum,and subcultured the mutant strains to structure the mutant library.Though the structuring of REMI mutant library of Dictyostelium discoideum,we picked 95 mutant strains(number:M1-M95).②Screening mutant strains of developmental defect of Dictyostelium discoideumBase on the mutant library of Dictyostelium discoideum, wild type of Dictyostelium discoideum AX2 and mutant strains(M1-M95) were chosen to screen the mutant strains of developmental defect of Dictyostelium discoideum though the development experiment.We screened 71 mutant strains to be abnormal from wild type which was about the duration of time of every stage of development process. ThRoughout the development process of these five stages "cell-aggregation-mound-slug-spore", there were 52 mutant strains which had two abnormal development stages, and there were 9 mutant strains which had three abnormal development stages, and there were 10 mutant strains which had four abnormal development stages. In addition there were 9 mutant strains which occured abnormity in the cell stage, and there were 20 mutant strains occured abnormity in aggregation stage, and there were 68 mutant strains which occured abnormity in mound stage, and there were 71 mutant strains which occured abnormity in slug stage. Abnormalities in the development process of mutant strains, mutant strains M26 occured development block after slug stage, and compared with the same group of wild type didn’t occured spore in the 24 hours of development time, and after a prolonged development to 30 hours, it has not entered the spore stage. After analysis, we thought the M26 was developmental defect of mutant strain.③Screening mutant strains of electrotaxis of Dictyostelium discoideumBase on the mutant library of Dictyostelium discoideum, wild type of Dictyostelium discoideum AX2 and mutant strains(M1-M95) were chosen to screen the mutant strains of electrotaxis of Dictyostelium discoideum though the electrotaxis experiment. After starving the cell for 2.5hours, the wild type cell and the mutant strain cell were put in the one and the same chamber with the electrotaxis experiment together. Through the electrotaxis experiment,we get the “directedness”, “displacement speed”, “track speed” and “persistency”which are four parameters of electrotaxis for statistical analysis. Through analysis, the electrotaxis parameters of the mutant strains M1-M95 don’t show significant difference compared with wild type,and we didn’t screen out the mutant strains of electrotaxis of Dictyostelium discoideum from REMI mutant library of Dictyostelium discoideum.