| Brine shrimp (Artemia) is a worldwide-distributed crustacean, widely used as a main food resource in aquaculture and applied in basic research areas ranging from developmental biology to evolution and ecology. Artemia presents interesting features in development and depending on the environmental condition, two developmental pathways can be taken namely ovoviviparous and oviparous development. In the former case, free-swimming nauplii are released from females and in another style encysted gastrulae (cysts) are produced under unfavorable environmental conditions. The cysts enter diapause, a state at which development arrests, metabolic activity reduces greatly, and the resistance to severe physiological stress is high. Upon breakage of diapause and placed in suitable conditions, the embryo is activated and resumes its development, releasing swimming nauplii. This is a special strategy of the brine shrimp to cope with harsh environment and to breed offspring. Therefore, the encysted diapause state of the brine shrimp embryo is a crucial feature and of major importance in Artemia's life history. Moreover, Artemia can withstand great physiological stresses. They can survive long term anoxia, temperature extremes, repeated hydration and dehydration, desiccation, and exposure to pollutants. Therefore, the brine shrimp is a good model for studies on the mechanism of stress resistance.In this thesis, the protein expression of the diapause, activated cyst and cysts at different postdiapause developmental stages were studied using proteomic methods. The changes in protein expression in response to CuSO4 in Artemia nauplii were also analyzed. A key genes of antioxidant enzymes: peroxiredoxin has been clone and analyzed. The main results are: A proteomic reference map for the diapause embryo of Artemia sinica was obtained using two-dimensional gel electrophoresis with a pH range of 4–7 and a molecular weight range of 10-100 kDa. Approximately 233 proteins were detected and 60 of them were analyzed by capillary liquid chromatography tandem mass spectrometry (LC–MS/MS). Of these, 48 spots were identified, which are categorized into 9 functional groups including cell defense, cell structure, metabolism, protein synthesis, proteolysis, and other processes.The protein expression profiles in the different postdiapause embryo development stages were detected and analyzed. The result showed that the activation stimulation induces apparent changes in protein expression in the activated cysts. Compared to diapause cysts, more protein spots were detected in the activated cysts proteome map, especially in the pH<5.5 range. There were approximately 70 protein spots up-regulated, including proteins unique in activated cysts; 25 protein spots down-regulated, including proteins unique in diapause cysts; and others, about 60%(in diapause cysts) expressed constantly after activation. The regulated proteins include heat shock proteins, anti-oxidant proteins and many other proteins. Isoforms of small heat shock protein p26, small heat shock protein ArHsp21 and peroxiredoxin expressed in activated cysts specifically.The protein expression at different stages in the hatching process showed different characteristics. We found 267,285,195 and 210 protein spots in the proteome map of 6h,12h,18h and 24h post hatching cysts. The cysts at 6h and 12h contain more protein spots than that of 18h and 24h. These changes could be attributed to the morphogenetic events and development process during the embryo development.Proteomic approaches were also applied to reveal the differential protein expression profiles of the acute responses to copper sulfate exposure in the larvae of Artemia sinica, which is a representative of high saline ecosystem. Fourteen significantly differentially-expressed proteins were detected and seven of these were determined through MS analyses. Three spots were up-regulated and identified as actin, heat shock protein 70 and chaperone subunit 1. Three down-regulated proteins were identified to be arginine kinase, elongation factor-2, and glycine-rich protein. An induced protein was identified as Peroxiredoxin (Prx). A Prx gene was cloned from nauplii of Artemia sinica by RACE and degenerate primers designed according to the peptide sequence noted by MS analysis and sequence homolog with Prx genes from other species. The full-length cDNA of Prx consists of 756 bp with a 594 bp open reading frame, encoding 198 amino acids. The molecular mass of the deduced amino acid is 22.0 Da with an estimated pI of 6.98. Sequence comparison showed that Prx of Artemia sinica shares 98% and 94% identity with that of A. franciscana and F. chinensis, respectively. Real-time PCR analysis indicated that the Prx gene transcripts increased to 3.0 fold of nomal level in nauplii of brine shrimp after exposed to CuSO4 for 24h. |
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