Under Cadmium Stress Blue Clams Antioxidant Enzyme Activity And Immune Gene Cloning And Expression Of Research

Cyclina sinensis is one of the major developing commercial shellfish in Chinese aquaculture. The healthy development of our county’s coastal breeding aquaculture was being deteriorated by the unceasingly expands of heavy metal pollution. Which has brought about the shellfish food safety problems was received the attention from the whole society. By the existing domestic and foreign relevant literature information retrieval, we found that has some reports of heavy metals in C.sinensis body and poison of accumulation, however, which related to C.sinensis immune response with the stress of heavy metal was rarely.In this study, on the basis of C.sinensis’s stress reaction against the adversity by the heavy metal pollution and C. sinensis immune enzymatic activity under Cd+stress, with the SMART method to built the clams C. sinensis cDNA library, then we using large scale EST sequencing and bioinformatics analysis methods to screen out immune gene sequences which related closely with heavy metal induced. After analysising we got the full length of C. sinensis MT and Trx cDNA. After analysised the two immune gene sequence characteristics, protein spatial structure, signal recognition mode and metabolic pathway, we analysed the expression of target gene in mRNA level with Real-Time PCR in different organizations, we may explain and gave some valuable experimental data about clam immune mechanism and immune transduction way.In this reaserch we established the experimental group and control group in a temporary condition, then we do the timing detection of the change of superoxide dismutase、catalase and glutathione peroxidase activity in different organizations when the clam was stimulated by Cd2+stress. After the stress test results showed the whole change trend of SOD、CAT、GPx activity is similar, first increased and then drop in blood and organ. The SOD in blood activity significant increased under Cd2+stress, reached its highest level at12h; The SOD in organ activity under Cd2+stress reached its highest level at24h; which significantly compared with the control group (P<0.05). And the activity decreased prolonged, however both at96h significantly compared with the control group (P<0.05). The CAT in blood and organ under Cd2+stress activity both reached their highest level at12h, which significantly compared with the control group (P<0.01) and then decreased. The GPx in blood activity under Cd2+stress reached its highest level at24h, which both at12h and24h significantly compared with the control group (P<0.01). The GPx in organ activity under Cd2+stress reached its highest level at12h, which significantly compared with the control group (P<0.05), and at96h significantly lower than the control group (P<0.05). There was a preliminarily evidence that the suspected Cd2+had significant effects on the three enzymes, and the activity was crimped after stress time more than a range.The researchers first built SMART-cDNA library of clam, then got the immune correlation factor gene sequences by large scale EST sequencing and bioinformatics analysis methods, and selected the metallothionein (MT) gene and thioredoxin (Trx) gene, then analyzed their full-length cDNA sequences. The results show that the full length cDNA of metallothionein and thioredoxin was776bp and804bp respectively. The open reading frame (ORF) of metallothionein which the length is225bp encoded74amino acids (aa) with7typical sequence:CXC, and there is a typical MT domain. The theoretical molecular weight of MT is7.18kD and theoretical isoelectric point is7.85. The ORF of thioredoxin which the length is498bp encoded165amino acids with a typical active position (Trp-Cys-Gly-Pro-Cys.), and there is a typical Trx domain. The theoretical molecular weight of Trx is18.75kD and theoretical isoelectric point is8.65; The space structure of Trx was predicted, which is the same as Ruditapes philippinarum’s. The registration number of Cyclina sinensis MT and Trx in the GenBank is HM246244and JN969050respectively.The researchers used real-time quantitative PCR (Real-Time PCR) to detect the timing expression characteristics of Cyclina sinensis MT and Trx gene in mRNA level in blood and liver under Cadmium (Cd). The results showed that that the expression of metallothionein gene significant increased under Cd2+stress from6h to12h, which was significant different from the control group (P<0.01), then it tend to normal level after24h. The expression level of thioredoxin gene increased under Cd2+intimidated at6h. And the expression of12h,24h was significant different from the control group (P<0.05), then continues for a longer time. Our investigation showed that Cd2+could induce the expression of metallothionein and thioredoxin gene, the transcription process of two genes reflects the molecular control process of the the shellfish against heavy metal stress, and the transcription reveals the characteristics of Cyclina sinensis MT and Trx gene expression to some degrees.After comprehensive analysised, we thought hat Cd2+has significant influence on C. sinensis’s immune disease resistance system, which infection could induced C.sinensis’s antioxidant enzymes activity increased significantly. And the C.sinensis’s SOD、CAT、GPx activity in blood and liver under Cd2+stress was the most sensitive, therefore, the changing of antioxidant enzymes activity of C. sinensis organizations that could be a biological marker of heavy metal pollution in marine environment monitoring. The researchers found that Cd2+could induce the excess expression of C. sinensis MT and Trx gene, which including the Large-scale transcription of MT and Trx, and the expression of MT and Trx gene with the increase of the stress time rise in the short term, and that states C. sinensis MT and Trx play an important role in the immune defense against heavy metal adversity. In addition, we compared the two immune protein sequence of amino acids with different animals, then we found C. sinensis MT and Trx was closer to the homologous protein of vertebrates in the evolutionary.