Cloning?prokaryotic Expression And Expression Analysis Of Apoptosos Related Genes Caspase9 And BCL-2 From Ctenopharyngodon Idellus Exposed To Cadmium

Apoptosis is a physiological protective mechanism of fish under developing, it plays an important role in the growth and development of fish. In this study, RACE(rapid amplification of cDNA ends) technique was used to clone the full-length cDNA of apoptosos related genes Caspase9 and BCL-2 from Ctenopharyngodon idellus, and characterization analysis of the genes, ranking the alteration of Caspase9 and BCL-2 mRNA levels from liver exposed to cadmium by real-time PCR. Prokaryotic expression, recombinant protein purification and mass spectrometry identification of the genes were performed, and also study the antioxidant enzymes responses and histological changes in the liver of Ctenopharyngodon idella after exposure to cadmium.The C. idellus Caspase9 cDNA is 1716 bp in length, containing a 1308 bp ORF, peptided encod 435 amino acids, with a calculated molecular weight of 48.24 kDa and an isoelectric point of 6.44. The 5 'untranslated region and 3' untranslated region are 225 bp and 183 bp. It was predicted without signal peptide and transmembrane domain, N terminal contains typical Caspase recruitment domain(caspase recruitment domain CARD), a Caspase family P20(large subunit, P174-E304) domain and P10(small subunit, A347-S434) domain. The C. idellus Caspase9 histidine active site H237SAYDCCVVIILSHG251 and cysteine active site K291PKLFFIQACGG302 are located in the large subunit P20 domain. The phylogenetic tree indicated C. idellus Caspase9 and Gobiocypris rarus Caspase9 kinship most closely. Real-time PCR showed that cadmium can induce the expression of Caspase9 genes in the liver of C. idellus, with the Cd concentration and exposure time increased, the expression of Caspase9 significantly up-regulated(P<0.05). According to the cDNA sequence, construction pET28a-Caspase9 prokaryotic expression vector, transformed into E.coli(Escherichia coli) DE3, SDS-PAGE electrophoresis analysis showed that, IPTG can induce the expression of vector pET28a-Caspase9, recombinant protein molecular weight of about 50 kDa, and consistent with prediction. Purified recombinant protein successfully by Ni-NTA His?Bind Resin, the molecular weight of the recombinant protein is 48.95 kDa and isoelectric point is 6.17 by mass spectrometry, which consistent with the predicted results, the recombinant protein expression is correct.The C. idellus BCL-2 c DNA is 1436 bp in length, containing a 687 bp ORF, peptided encod 228 amino acids, with a calculated molecular weight of 26.19 kDa and an isoelectric point of 5.09. The 5 'untranslated region and 3' untranslated region are 236 bp and 513 bp. It was predicted without signal peptide, with a hydrophobic transmembrane domain(S204-A226) in the C-terminal. N-terminal contains typical BCL-2 homology domain BH4(N9-W28), but also includes BCL-2_like homology domains BH1, BH2 and BH3(L69-A191). The phylogenetic tree indicated C. idellus BCL-2 and Gobiocypris rarus BCL-2 kinship most closely. Real-time PCR showed that cadmium can effect the expression of BCL-2 genes in the liver of C. idellus. The expression of BCL-2 in the liver of C. idellus was significantly upregulated(P <0.05) when fish expose to 0.5mg/L Cadmium solution for 7 days. The expression of BCL-2 was inhibited in other treatments. According BCL-2 cDNA sequence, remove the transmembrane domain sequence to construct pET28a- expression vector, transformed into E. coli DE3 in, SDS-PAGE electrophoresis analysis showed that, IPTG can induce vector pET28a-BCL-2 expression, recombinant protein molecular weight about 28 kDa, consistent with the prediction. Purified recombinant protein successfully by Ni-NTA His?Bind Resin, the molecular weight of the recombinant protein is 22.77 kDa and isoelectric point is 4.88 by mass spectrometry, which consistent with the predicted results, the recombinant protein expression is correct.Cadmium stress can cause varying degrees of influence on the histological and antioxidant enzyme systems of C. idellus. Microstructure showed high concentrations of cadmium on C. idellus liver caused significant tissue damage, congestion of central vein, hepatocyte hypertrophy, nuclei changed, and the vacuoles in hepatocytes were observed. The activity of antioxidant enzymes in liver of C. idellus was changed after exposed to cadmium. Compared to control fish, the activity of superoxide dismutase(SOD) increased at 5?g Cd/L but significantly decreased at higher Cd concentration(P<0.05). Meanwhile, the activities of Catalase(CAT), Glutathione peroxidase(GPx) decreased significantly at concentrations of 50 and 500?g Cd/L(P<0.05), The level of Malondialdehyde(MDA), was up-regulated increasingly at higher concentration of waterborne cadmium(P <0.05).