Of Cytoskeletal Proteins Spiroplasma Their Cell Morphology Relationship

Eriocheir sinensis is an economically important species cultured in China in recent years. With the development of intensive aquaculture, various diseases have emerged, and started to threaten the sustainability of the aquaculture populations of E. sinensis, severely affected its production. The tremor disease (TD) is most devastating disease of E. sinensis. A kind of spiroplasma is identified as a novel causative pathogen of the disease and given the name of Spiroplasma eriocheiris. The biochemical and biological properties, evolutional analysis, identification of the pathogen as well as antibacterial tests of the spiroplasma has been studied, but the study on proteomics of the cytoskeleton has not been reported. Meanwhile, spiroplasma cytoskeleton has been international hotspot because of its unique characteristics. In the present study, we applied bioinformatics softwares to analysis the genes of cytoskeleton based on the complete genome sequence of S. eriocheiris and Spiroplasma mirum. Simultaneously, three cytoskeleton proteins had been detected by RealTime Polymerase Chain Reaction (RT-PCR), protein EF-Tu had been expressed in prokaryotic system and corresponding polyclonal antibody had been prepared successfully.1. Bioinformatics analysis of the cytoskeleton proteins in spiroplasmaSpiroplasma eriocheiris (S. eriocheiris) is the first spiroplasma found in fresh water crustacean. Its morphology changes greatly in every growing period due to lacking of cell wall, and it is inferred that these changes are probably caused by cytoskeleton. Therefore, in order to study the relationship of the cytoskeleton between S. eriocheiris and plant spiroplasma such as Spiroplasma citri, as well as insect spiroplasma(Spiroplasma mirum, Spiroplasma melliferum), we analysised the genes of cytoskeleton with bioinformatics softwares. The results show the following: (1) The base sequence similarities of four spiroplasmas were between 79%-98%; however, the amino acid sequence similarities changed greatly, which were between 33%-99%.(2) The results of SWISS MODEL showed that the same proteins from different spiroplasmas were nearly identical in three-dimensional structure. For example, the three-dimensional structure of EF-Tu (elongation factor Tu) has no difference in three spiroplasmas (S. eriocheiris; S.mirum; S. citri) and Richettsia, but is different to Mycoplasma pulmonis, E. coli, Cyanothece and Chlamydia trachomatis (Fig.2.1). However, the three-dimensional structure of Fib is nearly similar among S. eriocheiris, S. mirum and S. citri and the first two spiroplasmas have subtle difference with S. citri (Fig.2.2).2. RealTime Polymerase Chain Reaction (RT-PCR) test of cytoskeleton proteinsThe morphology of spiroplasma changed greatly when it growed in medium in different period of time. According to the new reports, the cytoskeleton of S. melliferum BC3 contained three components:Fib, EF-Tu and MreB. Therefore, the three genes were chosen to analyse their expression differences at every period and explore the relationship between cytoskeleton and morphology based on the complete genome sequence of S. eriocheiris and S. mirum, which form the foundation for the research of pathogenesis.The results suggested that:S. eriocheiris appeared spiral, rodlike and globularity at exponential phase, stationary phase and death phase respectively. The results of RT-PCR showed that the expression levels of cytoskeleton genes also declined gradually with these three phases, which was positively correlated with spiral shape. In the same way, S. mirum appeared small number of spirals at exponential phase and more number of spirals at stationary phase, and incompact banding at death phase. The results of RT-PCR showed that the expression levels of cytoskeleton genes also positively correlated with spiral shape. In conclusion, it could be inferred that three cytoskeleton genes (Fib, EF-Tu and MreB) may play an important role in cell morphology. 3. Clone, expression and function study of cytoskeleton protein EF-Tu (elongation factor) from S.eriocheirisEF-Tu gene was first obtained from S.eriocheiris in this study. First, partial sequence of EF-Tu gene was cloned, linked with plasmid pET28, and transduced into E. coli for reproduction. Second, in order to prove that protein EF-Tu we acquired had immunogenicity, we expressed the recombination protein EF-Tu-His by prokaryotic expression system, and purified it with Ni-NTA chelating affinity chromatography, identified by mass spectrum and prepared rabbit polyclonal antibody also. Third, the results of SDS-PAGE and Western Blotting made us believe that the protein was expressed successfully and had immunogenicity. In order to verify whether the protein was from S. eriocheiris, we isolated total protein of three phases from S.eriocheiris, and detected it by polyclonal antibody. The results of Western Blotting suggested that total protein were reacted with polyclonal antibody, and the immune banding was found at 43KDa, which was the same as expected.Western Blotting was prepared to study the differences of expression levels at three phases. The results showed that the expression level of protein EF-Tu was gradually increased with exponential phase, stationary phase and death phase, which was negatively correlated with spiral shape. Three reasons were speculated:(1) EF-Tu was multifunction protein, and it distributed in cytoplasm to assist linking and sustaining. Its position changed from cytoskeleton to elongation factor while bacterium growed. (2) There were many regulatory factors from transcription of genes to synthetize of proteins, which may result in different consequences at gene and protein expression levels. The study provided important theoretical foundation for studying the disease control and immunologic mechanism between the antigenic proteins and hosts.