| This topic focused on studying the mechanism of protozoan dormancy. Euplotes encysticus was used as the experiment material, which is one kind of ciliates. It has all the characteristic of eukaryotic cells, studying the nature of its encystment process can provide a theoretical basis for the regulatory mechanism of eukaryotic cell differentiation. This study analysised the difference of protein and gene expression by iTRAQ technology and real-time fluorescent quantitative PCR(qRT-PCR) technology. At the same time, FLUTAX (paclitaxel) direct fluorescence labeling technology and Phasecontrast microscope technology were used to analyze and compare the morphology of resting cell and vegetative cell; And the function of P-tubulin gene in vegetative cell was studied through RNA interference technique. Finally, the composition of cyst wall was identified by LC-MS/MS technology.Using iTRAQ teachnology to analysis and identify the difference proteins between resting cell and vegetative cell, found out 21 proteins related to encystment.Compared to vegetative cells, there were 9 up-regulated proteins and 12 down-regulated proteins in resting cells. These different proteins involved in many cell life activities, including the structure of cytoskeleton, gene expression, the synthesis and metabolism of energy and stress reaction, etc.; The result also found out 10 kinds of proteins without the ratio of 115/114, which might be the specificity proteins in resting cell or vegetative cell. Then we found that cilia largely disappeared, only retained part of the cilia during the encystment process, which verified that tubulin proteins really changed significantly in the process.The relative quantitative of some genes related to encystment were analyzed by qRT-PCR technology. There were 16 differential expression genes in resting cells which compared to vegetative cells,10 out of them were obviously down-regulated, the other 6 genes were up-regulated significantly. These differentially expressed genes play important roles in the process of cell differentiation regulation, and make the vegetative cell can transform into cyst smoothly, so as to survive in bad environment. The expression of some genes associated with energy metabolism were lowered.The respiration intensity of resting cells were also weaker than vegetative cell detected by cell respiration metabolism detection kits. These results indicated that the life activities of resting cells greatly abate, only maintained some basic activities to survive.Significant changes have taken place in the inside and outside cells after interfering β-tubulin gene of vegetative cell. The results of direct fluorescence labeling technology showed that the experimental group accumulated a large number of granular material in cells. Under the same experimental conditions, the longer the interfering time is, the more sensitive the cell is; the cellular membrane of the experimental group appeared rupture phenomenon, but the control group had no. The results show that the β-tubulin gene plays an important role in maintenance of cell shape and material transportation. The result of MTT showed that: in the beginning of 4 to 6 days, the cell growth of both experimental and control group was on the rise; Two days later, the experimental group growth decline, while the control group was kept steady growth; And then the cell growth all returned to rising trend, but the growth curve of the experimental group was located in the below of control group. It suggested that interference the β-tubulin gene will affect cell growth.While the expression of β-tubulin gene had no apparently different, showing that the interference had no obvious effect on the expression of the gene,it may affect the cell life activities and morphological structure through other ways.This study also made use of LC-MS/MS technology to separate and identify the composition of the cyst wall proteins, total identified 61 kinds of trusted proteins, including many kinds of tubulin proteins, transport proteins, signal transduction proteins and energy metabolism related enzymes. Then we also observed the surface morphology of cyst wall through Phasecontrast microscope technology and FLUTAX direct fluorescence labeling technology, and found that the surface has a lot of convex structure which composed of tubulin proteins.The study made a more comprehensive comparative analysis between resting cell and vegetive cell from the perspective of protein, gene, energy metabolism and so on.It provides important information for protozoa dormancy mechanism, and is very helpful to insight into the eukaryotic cells differentiated regulation mechanism under adversity. |
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