| Protist ciliates are a wide variety of eukaryotic unicellular animal. Due to their most complicated cellular differentiation, some ciliates have become the excellent study materials. Encystment and excystment not only exsit in parasitic protozoa, they are also relatively common phenomena in free-living ciliates. Under adverse environmental stresses, such as sudden changes of ambient temperature, the lack of food supply and high population density, ciliates can undergo complex life activities and many species of them will experience a differentiation process, changing from vegetative cell into resting cyst to withstand the infestations triggered by a variety of harmful factors in their surroundings. Excystment will occur when the external environmental conditions are suitable. It can be said that cyst formation is a superior strategy for ciliates to survive in stress conditions. Study on ciliates dormancy has become one of the most important aspects of researching the eukaryotic cell structure and mechanisms of functional regulation. At present, abundant information mainly focusing on microscopic and submicroscopic levels has been accumulated, which is associated with morphological and physiological changes of ciliates during encystment. These changes between resting cyst and vegetative cell indicate that there are a series of genes and proteins differentially expressed during the period of ciliates cell differentiation. Studies on the encystment mechanism of ciliates at protein and gene levels, however, are now quite limited. Study at molecular level might make us have a deeper understanding of protein changes and the mechanisms of gene regulation during encystment. Meanwhile, it also provides important molecular biology information to explore the mechanism of eukaryotic cells resisting adversity and interaction with their surrounding environment.In this article, Pseudourostyla cristata was selected as experimental material, proteomics technologies, real-time fluorescence quantitative PCR (qRT-PCR) and bioinformatics were applied to comparatively analyze resting cysts and vegetative cells at molecular level from multi-perspective, mainly focusing on the qualitative and quantitative analysis of differentially expressed genes and proteins in resting cysts and vegetative cells of Pseudourostyla cristata. The protein components of cyst wall were also analyzed and identified. Dependent on the comparatively studies of resting cysts and vegetative cells of Pseudourostyla cristata at molecular level, a lot of genes and proteins involved in dormancy were screened and identified, the functions and roles of the dormancy-related genes and proteins were revealed in this study. The results and conclusions are as follows:1. Proteins identification of resting cysts and vegetative cells of Pseudourostyla cristata by LC-MS/MSThe total proteins extracted from resting cysts and vegetative cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) electrophoresis and identified by liquid chromatography with tandem mass spectrometric(LC-MS/MS) technology. Subsequently, bioinformatics were used to further annotate the identified proteins functions.sThe identified results of LC-MS/MS showed that 668 protein groups were detected in resting cysts and 102 high credible proteins were included, while 88 high credible proteins out of the 724 protein groups were found in vegetative cells. Respectively,102 and 88 high credible proteins in resting cysts and vegetative cells were selected to be comparatively analyzed. The result showed that the majority of all high credible proteins we detected were common-expressed both in resting cysts and vegetative cells, whereas each of them had very few specific proteins. There were also some uncharacterized proteins in our results. The identification of these differential proteins suggested that changes of protein expression existed in the process of vegetative cells transforming into resting cysts. Understanding and analyzing the functions of these common-expressed and specific proteins may not only help to explore their roles played in the process of cyst formation, but make important significance in revealing the molecular mechanism of ciliates encystment as well.2. Analysis of differentially expressed genes involved in dormancy of Pseudourostyla cristataBased on identification and analysis of the proteins in resting cysts and vegetative cells by LC-MS/MS, a part of high credible proteins were selected to design their gene primers. Using real-time fluorescence quantitative PCR (qRT-PCR) technology, expression levels of these genes in resting cysts and vegetative cells of Pseudourostyla cristata were compared and analyzed. In the selected 47 genes,27 genes were efficiently amplified. Of which 20 genes were up-regulated, including calcium channel gene, calmodulin gene and 26S proteasome gene, etc; 7 genes including cathepsin B gene, rRNA gene and a-tubulin gene were down-regulated. So many changes in gene expression showed that encystment of Pseudourostyla cristata was a process of multi-genes collaborative regulation. The analysis of these changes on gene expression levels not only helped us to understand the roles of these genes in Pseudourostyla cristata during ensytment, but also provided us with the regulation of gene expression involved in the process of cyst formation.3. Identification and analysis of cyst wall proteins of Pseudourostyla cristataIsolation and identification of the cyst wall proteins of Pseudourostyla cristata was one of the highlights in this study. Cyst wall proteins were separated by applying SDS-PAGE electrophoresis, mass spectrometry was also used to analyze and identify eight protein bands of electrophoretic separation. The identified results were mainly the stress protein and structural proteins, including HSP70, β-tubulin, beta-1,3-N-acetyl galactosaminyl transferase, Leucine rich repeat family protein as well as some hypothetical proteins. Subsequently, we analyzed the functions of these identified proteins. The results of this study provided important molecular biology information to elucidate the components of cyst wall proteins of Pseudourostyla cristata.This study explored the protein events of cell differentiation and mechanisms of gene regulation at protein and gene levels, which provided us significant information of the inherent regulatory mechanisms of cells differentiation transferring vegetative cells into resting cysts of ciliates and the mechanism of cellular resistance to adversity in eukaryotic cells. |
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