| The swimming crab, Portunus trituberculatus (Crustacea: Decapoda: Portunidae: Portunus) is a commercially important species, widely distributed along the coast of China. It is a euryhaline crab species and can survive in wide-range salinity conditions, but different water salinity conditions might influence its distribution and migration rout. The swimming crab natural resources were declinied significantly due to overfishing in the past several decades. The artificial propagation of this species was promoted along the coastal waters, and now the swimming crab became the most important farmed marine crab in China. Salinity is an important factor in the crab artificial propagation. There were lots of literatures regarding on the osmotic regulation physiological mechanisms of euryhaline crab so far, while the researches on molecular mechanism was quite few. To clarify the molecular mechanism of osmotic regulation has great significance on the improvement of germplasm resources and artificial propagation of P.trituberculatus. The screening of osmo-regulation related genes is the necessary prerequisite of the study on the mechanism of osmotic regulation.In our study, full-length cDNA library was constructed from gills of the swimming crab, Portunus trituberculatus. 5012 clones randomly picked out from cDNA library for sequencing, resulted in 4433 high quality ESTs with the average length of 748bp. All the ESTs were assembled using CAP3 software,producing 2426 potential transcripts (238 contigs and 2188 singlets). The number of ESTs per contig is between 2 to 641, with an average of 10.34 ESTs. The length of contigs is between 293 and 1356bp, with an average length of 796bp. All transcripts were compared to non-redundant protein database of NCBI using BLASTx, and homologous sequences can be found for 858 ESTs(E-value<10?4).The results of primary annotation indicated that protease (1.7%), ribosomal protein (5.2%) and mitochondrium (6.9%). Similar sequences were classified by species, we found that Drosophila melanogaster (20.05%), Mus musculus (8.51%), Rattus norvegicus (8.04%), Homo sapiens (7.58%), Xenopus aevis (3.96%) and Danio rerio (3.96%). The functional annotation of the P. trituberculatus transcripts resulted in identification of about 292 unique sequences (629 ESTs) involved in salinity adaptation, accounted for about 12.0% (292/2426) of total unique sequences and 14.2% (629/4433) of the total ESTs. Some of them were identified according to GO classifications; others were determined based on the published literature on the genes related to salinity adaptation. These candidate genes were further grouped into at least seven function categories: reactive oxygen scavengers (5.4%), transporter protein (15.8%), stress protein (11.0%), signaling transduction (13.7%), protein synthesis and destination (22.4%), transcription (13.7%), and metabolism and energy (17.5%).By using T3 and T7 primers, 2426 uni-genes were amplified by PCR and the purified PCR amplification products were used for preparation of cDNA array (CapitalBio Corporation). Each product was repeated three times on the array. Total RNA Were extracted from three groups of samples (10‰, 25‰, 40‰) and named as G-10, G-25 and G-40 respectively. Total RNA was used to prepare the fluorescent dye labeled cDNA using the linear mRNA amplification procedures were performed by CapitalBio Corporation. Then the RNA samples of treatment and control were labeled with either fluorochromes cyanine-3 (Cy3)-dCTP or cyanine-5 (Cy5)-dCTP. Analyses were performed twice per sample, using a dye-swapping experiment (technical replicate) in which cDNA from the control (G-25) was labeled with Cy3 and cDNA from low salinity (G-10) or high salinity (G-40) challenge was labeled with Cy5. In the second analysis, control cDNA was labeled with Cy5 and cDNA from salinity challenge was labeled with Cy3. This dye reversal helps to minimize error due to fluor-associated bias. Therefore, we could obtain differentially expressed genes between low-salinity/high salility and control conditions separately.We obtained a total of 417 significantly differentially expressed genes by cDNA microarray screening. The differentially expressed genes were grouped using hierarchical cluster analysis, and eight different clusters were defined. Each Cluster has different gene expression profile.The response to low salinity (10‰) was a dramatic up-regulation of 158 genes and a down regulation of 54 genes. And the response to high salinity (40‰) was a dramatic up-regulation of 123 genes and down regulation of 222 genes. Interestingly, 5 genes showed concomitant up-regulation whereas 10 down-regulation in both low and high salinity challenges. 130 genes showed opposite direction of regulation responding to the two different salinity challenges.The expression levels of 14 genes were analyzed by real time quantitative RT-PCR at different salinity conditions (10‰, 25‰, 40‰) to demonstrate the results of array hybridization. The results indicated that 12 of 14 genes were consistent with array analyzed. |
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