| Stylonychia is a kind of common freshwater ciliate, widely distributed in the abundant aquatic plants ponds, rivers and swampy waters. As a kind of convenient collection of unicellular eukaryotes, sensitive to the change of the environment, and easy to culture, is a good material for research of life an important feature of eukaryotes.Epidermal growth factor EGF, is a kind of small peptide composed of 53 amino acid residues, a multifunctional growth factor, has found it could improve the activity of DNA topoisomerase in cells, and promote the expression of some genes associated with proliferative function. In vivo and in a variety of tissues and cells have strong mitogenic effect.In this study, the Stylonychia lemnae as the research object, based on the growth rule, exposing them to a certain concentration of EGF, to observe the effect of EGF on the production of Stylonychia, and by transcriptome sequencing method, analysis of gene in Stylonychia EGF exposure and different growth stages. The expression at the genomic level, discusses the effects of EGF on the molecular mechanism of stylonychia.The results are as follows:1 Established the growth curve of Stylonychia. Cultivation of cells in 24 well plate,set up different inoculation density counted regularly, after repeated experiments, the calculation of the cells under this system the generation time is about 18 h. And found the best density in 24 inoculation, the inoculum density is 30cells/ml, the 96 h division of the fastest, density is 1500-2500cells/ml, to 108 h when the number reached a peak,the density of 4000-5000cells/ml, 120 h into the decline phase density is about1200cells/ml. The growth trend of the most stable, the most suitable growth period, to observe and study the influence of Stylonychia proliferation and EGF production of Stylonychia.2 Found that the EGF has a promoting effect on the separatist. With different concentrations of EGF in Stylonychia, found in the concentration of 10 u g/ml EGF, the cells proliferation rate was significantly increased when the inoculation density:30cells/ml, the calculation results of 19 h in the control group and the experimental group generation, the generation time is shortened to 17 h, and in the logarithmic phase,the platform period and decline the cell density was higher than the control group.3 Discusses the role of EGF gene in Stylonychia mechanism. Select 96h(logarithmic), 108h(platform) and 120h(decline) cell transcriptome sequencing,molecular mechanism of action of EGF to promote the proliferation of stylonychia. First characteristics from gene level to understand Stylonychia growth trend in each period.The results show that: the logarithmic growth phase, due to the effects of EGF high expression genes included in the transcription process, nucleosome assembly,chromosome organization and protein folding in the process of synthesis, the period of the most exuberant vitality of cells in each cell material. The platform stage, protein folding, biosynthesis, redox process, coenzyme metabolism, glucose metabolism, cell metabolism of ketone, ethanol metabolism process. The maximum cell density cell platform, feel the lack of space, breathing, p H pressure, in order to adapt to the environment will have some changes; the decline phase, the difference of gene enrichment in DNA, metabolism, binding to tubulin, microtubule cytoskeleton, stress response, DNA repair, DNA conformational change, at this time, the number of accumulation. Metabolite accumulation is greater than the number of divisions of death.On this basis to further explore the molecular mechanism of EGF on Stylonychia proliferation effect and EGF effect in Stylonychia. Found a number of biological processes and pathways regulated by EGF, such as signal transduction, calcium ion transport, MAPK signaling pathway, calcium signal transduction pathway, P53 signaling pathway. These new findings explain the molecular mechanism of EGF promoting the spiny tailed second division, but also provides a new clue for the study of Stylonychia and EGF. |
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