Study On The Dormancy Molecular Mechanism Of Hypotrichida Ciliate, Euplotes Encysticus

The protozoan ciliate is a kind of eukaryotes with high degree of differentiation. There will be a special physiological state in the life of ciliates when it encounters unfavorable growth factors, such as lack of food, too intensive of polypide, changes of extreme temperature. Ciliate can take place a series of physiological and morphological changes. For example, cells becoming smaller, changing to round, forming cyst wall through secrete special substances. It will become excystment when the environmental conditions are favorable. Such survival mechanism is related to their mechanism of dormancy. Ciliates dormancy mechanism has become an important content of the research on ciliates’s system biology and developmental biology. Current research on the ciliate resting phenomenon, both domestic and international, are most focused on cell microstructure level and cell differentiation of morphological changes. Study on the mechanism of regulation on the level of biochemical and molecular can better explain the cells differentiation during the time.We used Euplotes encysticus as our experimental materials which is a kind of hypotrichida ciliate that is easy to culture and induce cell differentiation. We studied differentially expressed genes and proteins, the composition of the cyst wall proteins with the real-time quantitative polymerase chain reaction techniques (RT-qPCR), two-dimensional gel electrophoresis(2-DE), and mass spectrometry technology (MS) and also bio-informatics technology between the cyst cell and vegetable cell. We studied the ciliate of Euplotes encysticus cell differentiation and regulation mechanisms at the level of gene and protein. The research results can help us to understand the nature of ciliates dormancy. It also provide important information for exploring eukaryotic cells how to regulation cell differentiation when resist adversity, and can clarify the characteristics and laws of the dormant cell. The obtained experimental results and conclusions are as follows:1. Screening and analysis of differentially expressed genes in the encystment of Euplotes encysticusDifferentially expressed genes between the cyst cells and vegetative cells of Euplotes encysticus were screened by using real-time quantitative PCR technique. These differentially expressed genes were following sequenced and comparativelly analysed. The results showed that the effective amplification genes were 52 which selected from 128 candidate genes. That is to say, compared to vegetative cells, we screened 52 differentially expressed genes including 24 up-regulated and 28 down. Levels of these genes differentially expressed were ranging from several times to several hundred times. Further gene sequencing analysis study showed that these genes which were 2 times of differentially expressed were the genes relateing to cell structure, signal transduction, growth and metabolism, stress response. The results suggested that the process of cell differentiation is a fine regulation by multiple genes.2. Analysis of differentially expressed proteins of Euplotes encysticus by two-dimensional electrophoresisWe analyzed the differentially expressed proteins between the resting cell and vegetative cells of Euplotes encysticus by 2-DE proteomic technology. Total of 26 protein spots were screened by comparing the two sets of experiment electrophoresis gel images.14 protein spots were detected from the two groups that are differentially expressed,12 protein spots were only exist in the cyst cell which was a new expression. Compared to vegetative cells,14 protein spots which acquired had 7 up-regulated and 7 down. The subsequent identification of newly expressed proteins found that serine acetyltransferase and trimethyllysine hydroxylase had an important role in cell metabolism and stress response under the adversity, the other protein spots were the proteins of unknown and not explicitly functional proteins. The results illustrated that the protein levels had changed during the cell differentiation. These different expression proteins had a combined effect on ciliate, and induced its from active to hibernation.3. Separation and identification cyst wall proteins of Euplotes encysticusThe cyst wall protein which was segregated by cell ultrasonication were investigated by SDS-PAGE, MS, bioinformatics techniques. The results illustrated that the cyst wall proteins were a complex which contained a variety of different proteins. These proteins are mainly related to the following:protecting cell, maintaining cell structure,composition of the membrane proteins, signal transduction, transmembrane transport and secretion,et al. In order to adapt to the adverse environment, the cells formed a wall that composed of a variety of functional proteins and carbohydrate. It provides a structural basis for the cells to withstand adversity.In summary, the results of this study will help us to understand the nature of ciliates dormancy, and provide the important information for exploring eukaryotic cells resist adversity regulation mechanisms and cell differentiation regulation mechanisms, clarify the characteristics and laws of dormant cells life activities.