| The brine shrimp, Artemia, is a small crustacean and found in a variety of harsh environment in which it must endure severe hypersalinity, high does of ultraviolet radiation, very low oxygen tensions and extreme temperature. Artemia is also a very important food resource in aqualculture, as well as plays a significant role in environment protection and monitoring, salt manufacture. Therefore, Artemia was extensively studied as a model animal recently, these studies focused on its biology, physiology, biochemistry, genetics, cellular biology, hatching and culture. Compared with other model animals, there are few reports on genetics and genomics of Artemia. Artemin is a member of the ferritin superfamily and thought to be a stress protein relating to the stress response of Artemia. But there is little information on it. In this context, we preliminary established an EST (expressed sequence tag) database from emerged larvae (EII) of Artemia franciscana by partial, single pass sequencing to cDNA clones, provided a powerful information resource of molecular biological studies on Artemia and filled the blank in the field of genetics and genomics research. Moreover, we firstly cloned and sequenced the cDNA of artemin and ferritin from Artemia franciscana, and characterized the molecular structure of these two protein as well as their expression patterns in different developmental stage. These results provided an important evidence for studies the functions of ferritin and artemin, and the mechanism of diapause and resistant to stress. The main results of this study are as below: 1. Establishment of EST database of Artemia franciscana (EII). We constructed a cDNA library from Artemia franciscana (EII) and obtained 609 cDNA sequences by partial and single-pass sequencing to 672 cDNA clones.a. The result of sequence homology searching revealed that there are 350 with significant similarity to database sequences of known function of which 78 matched ribosome RNAs. Twenty sequences matched hypothetical open reading frames of unknown function, while!61 clones did not match any sequences in the database and presumably represented previously unidentified cDNAs. 350 known sequences belong to 219 different genes, 14 genes were identified previously in Artemia and 215 are firstly discoveredb. 448 ESTs were separated into 4 classes according to their cluster size: the first class is highly expressed sequence, such as mitochondrial 16S ribosomal RNA; the secondary class is medium size (>10), including cytochrome b and cytochromeoxidase C subunit I; the third one is low prevalence containing 63 different genes; the last is the ESTs with single copy.c. Anemia ESTs encoding identified proteins were classified into 8 functional categories: cell division, cell structure and motility, metabolism, signal transduction, cell defense, protein/gene expression, miscellaneous and hypothetical protein. There are a large number of ESTs which encoding protein relating to metabolism and protein/gene expression.d. Statistical analysis of codon usage and GC content in 228 ESTs encoding known function protein and hypothetical protein, and the comparison with human and fruitfly revealed that there is a significant difference in codon preference, GC content among these three species, especial GC content in the third position of each codon.2. Cloning and molecular characterization of ferritin and artemin cDNA from Artemiafranciscana.The cDNA of ferritin and artemin were cloned first time and sequenced in full-length.a. The artemin cDNA (accession number, AY062896) is composed of 2,072 bp containing a 690 bp ORF, flanked by a 25 bp 5'-UTR and a 3'-UTR of 1,357 bp with poly(A) tail. Two poly- adenylation of AATAAA are located within the 3'-UTR at nucleotides 1087-1092 and 2035-2040 respectively. A GT box of 18 consecutive nucleotides required for efficient processing and polyadenylation of mRNA appears at position 885-902. Moreover, there are two motifs of "ATTTA" and four variants of "ATTTTA" relating to the stability of...
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