| The ciliated protozoan is a kind of unicellular eukaryotes. When the environment is detrimental to its vital movement, it will usually form a resting cyst through secreting special substance, and enter into the dormant condition. If the surroundings then turn to be benefit for its vital movement, it will go through excystment and become a vegetative cell again. Some researches show that ciliate will differentiate and regulate itself in a way different from the vegetative cell during encystment and excystment, which makes the resting cell become a special study model. The study on ciliate's encystment has been a significant aspect of the research on ciliate's systematology, cyto-architecture, cell function, the formation of cell model and regulating mechanism. So far, the researches on encystment mainly focused on the following aspects:the formation of cyst's structure, and the changes of cytoplasm and nucleus at microscopic and ultramicroscopic level during encystment; the dedifferentiation and differentiation of cell cortex cilia, and so on. Only very limited information is available about the modulation of gene activity from a vegetative cell to a resting cyst. Study on encystment at biochemical and molecular level can be significantly helpful for us to more deeply understand the essence and modulating mechanism of encystment.Based on the former achievements, using Pseudourostyla cristata and Urostyla grandis as research objects, mainly utilizing real-time fluorescence quantitative PCR (RT-PCR), two-dimensional electrophoresis, mass spectrometry and bioinformatics, from a multitude of perspectives, we qualitatively and quantitatively analyze genes and proteins in resting cysts and vegetative cells of the two hypotrichous ciliates. The purpose is to find out these genes probably related to the regulation progress from vegetative state to dormant state, and the protein change. The results are significantly meaningful in these research fields:the study on the material basis of life activity and their possible function; the research on the structure and function, the metabolism and regulation of eukaryocyte in dormant state; the study on cell's characteristics of life activities and law of life. The followings are results and discussions.1.Relative quantitative analysis on gene expression level between resting cysts and vegetative cells of Pseudourostyla cristataWe applied RT-PCR to analyze some genes' expression level resting cysts and vegetative cells of Pseudourostyla cristata. In the later stage, we sequenced the production of PCR, and then utilized BLASTx and BALSTn in NCBI to compare them with protein and nucleic acid which are known for their' sequences and function. Based on the information, we discussed their possible function during encystment. The results are that in the fifteen RT-PCR with fifteen different special primers there are DNA production, but the amounts in resting cysts and vegetative cells are different, which has been confirmed by several times repeated experiments. Moreover, the expression level of one gene decreases in resting cyst, while that of others increases. The results show that there is difference in gene expression level between resting cysts and vegetative cells of Pseudourostyla cristata. Therefore, we speculate that there genes are necessary for ciliate's encystment.2.Relative quantitative analysis on gene copy number between resting cysts and vegetative cells of Urostyla grandsWe applied RT-PCR to analyze some genes' copy number between resting cysts and vegetative cells of Urosty/a grandis. In the later stage, we sequenced the production of PCR, and then utilized BLASTx and BALSTn in NCBI to compare them with protein and nucleic acid which are known for their' sequences and function. Based on the information, we discussed their possible function during encystment. The results are that in the seven RT-PCR with seven different special primers there are DNA production, but the amounts in resting cysts and vegetative cells are different, and the copy number of the seven genes all decrease in resting cysts, which has been confirmed by several times repeated experiments.The results show that there are difference in gene copy number between resting cysts and vegetative cells of Urostyla grandis, and the expression level can be influenced by the gene copy number to some degree. Therefore, we come to the conclusion that the gene copy number is probably related to ciliate's encystment by influencing genes'expression level.3. Two-dimensional electrophoresis analysis and mass spectral analysis on holoprotein between resting cysts and vegetative cells of Urostyla grandisWe applied two-dimensional electrophoresis, mass spectrometry and bioinformatics, which are the key techniques of proteomics, to comparatively analyze holoprotein between resting cysts and vegetative cells of Urostyla grandis. After comparing the two-dimensional electrophoresis maps, we selected out32protein spots of which the quantity variance is more than3times. Among these spots,20protein spots are up-regulated and12protein spots are down-regulated in resting cysts. Then we selected6protein spots to do mass spectrometry, and found out the known proteins which comparatively best matched these proteins. These known proteins were amino acid ABC transporter, thermostable serine protease and type Ⅱ keratin. We analyzed the probable function of these proteins during encystment based on this information. |
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