Study On The Effects Of Free Amino Acid Metabolic Pathways Of Portunus Trituberculatus Under Salinity Stress

The swimming crab, Portunus trituberculatus(Arthropoda, Crustacea, Decapoda, Portunus) was an important marine economy crab. As the predominant aquatic animal, crustaceans can dwell in seawater and fresh water due to their various osmoregulatory mechanisms. The two forms of osmoregulatory mechanisms were anisosmotic extracellular regulation(AER) and intracellular isosmotic regulation(IIR). AER mainly can control the osmolality of the body fluid through regulation of gills and antennal glands, while IIR maintained intracellular osmolality and maintain the balance between tissues and the hemolymph primarily by regulating free amino acids(FAAs).The study consists of three parts:The first part: Study on the effects of free amino acid metabolic pathways of Portunus trituberculatus under salinity stress.The highest levels was in muscle, followed by the gills and hemolymph. The total free amino acid, the content of total free amino acids in these tissues were positively associated with the outside salinity(P<0.05). The main osmoregulators were Ala, Glu, Arg, Gly and Pro. It was worth noting that Pro played a leading role in high salinity. Therefore, clear Pro metabolic pathways and genes in the role of gene under salinity stress were very important for P. trituberculatus.The second part: The effect of salinity stress on Glutamic acid dehydrogenase(GDH) and gene expression and activity in Portunus trituberculatus. The expression of high salt promoted the GDH and low salt inhibits the expression of GDH. The GDH activity changes were inverse to expression under salinity stress. So the experiment results show that synthesis Pro was mainly depend on by his metabolic pathways, rather than simply was controlled by GDH.The third part: firstly, Cloning of Ornithine Aminotransferase gene from P. trituberculatus and the effect of saliniy stresses on its gene expression and activity. The cDNA of OAT(Ornithine aminotransferase)was 1754 bp in length, which contained an open reading frame(ORF) of 1317 bp, encoding a 438 amino-acid polypeptide. Homology analysis revealed that the similarities of OAT of p. trituberculatus with the OAT of Metaseiulus_occidentalis was 74%. High salt and low salt stress significantly inhibit the expression of the OAT. The OAT activity changes were similar to expression under salinity stress. Maybe the Ornithine pathway was not the main way to synthesize Pro under salinity stress. Sencondly, the effect of salinity stress on Δ1-pyrroline-5-carboxylate synthetase(P5CS) and gene expression and activity in Portunus trituberculatus. There was no significant influence on the expression of P5 CS gene under salinity stress(P<0.05). while the P5 CS activity was activated in high salinity while was suppressed in low salinity. Therefore P5 CS played a main role for accumulating Pro under salinity stress. Namely Glu pathway was main way for synthesis of Pro. Thirdly, Cloning of Proline dehydrogenase gene from P. trituberculatus and the effect of saliniy stresses on its gene expression and activity. The cDNA of ProH was 2162 bp in length, which contained an open reading frame(ORF) of 1890 bp, encoding a 629 amino-acid polypeptide. Homology analysis revealed that the similarities of ProH of p. trituberculatus with the ProH of Stomoxys calcitrans were 62%. The expression of ProH gene was negative correlated with the outside salinity(P<0.05). ProH activity was activated in low salinity while was suppressed in high salinity, especially in 50 salinity. Therefore ProH played a significant role for Pro accumulation to swimming crab under salinity stress.