| Thioredoxin system is one of important antioxidant systems in organisms,which constitutes of thioredoxin (Trx), thioredoxin reduetase (TrxR) and NADPH.It plays a pivotal role in regulating enzyme activity, transcription factor, variousstress responses and signal transduction in organisms. Antarctica, due to itsspecific geogrpahical position and climate, is considered an exrteme environmenton the earth, and contains rich microbial resources. In the long process ofevolution and growth, Antarctic microorgnaisms have formed unique gene bank,genetic background and metabolic characteristics. Conseuqently, we selectAntarctic sea-ice bacteria Pseudoalteromonas sp. as research material, anddescribe the essential information of its thioredoxin system by cloning, expressingand analysing activity characteristics of thioredoxin and thioredoxin reductase.Therefore, we can further know the low-temperature adaptation mechanism ofAntarctic microorganisms, gain functional genes with independent intellectualproperty rights, and provide new resistance genes for biological genetic breeding.(1)On the basis of preliminary studies on cloning integral PsTrx gene fromPseudoalteromonas sp.ANT178, we used various bioinformatics methods toanalyse PsTrx gene sequence. The gene size was327bp, and it encoded108amino acids. The protein theoretical molecular weight was11.9kDa, and its pIwas4.50. We speculated that its key active sites were Cys33and Cys36, and itsstructure contained4α-helixs and5β-folds. The PsTrx gene was transfered intoE.coli BL21to realize heterologous expression. The Trx of Pseudoalteromonassp.ANT178was purified by Ni-affinity chromatography and measured proteinactivity. The result was showed that its specific activity was96.67U/mg and yieldreached27.26%. Its optimum temperature was25℃, and the optimum pH was7.0.The PsTrx in the high salt (2M NaCl) environment still retained some activity. Itwas inhibited by denaturant SDS strongly, and its relative activity was only22.94%. But it was little affected by the oxidant H2O2, and its relative activity wasup to95.36%.(2)After the PsTrx gene was taked into E.coli BL21, salt tolerance of therecombinant strain was improved significantly. In culture conditions of9%salinity,the recombinant strains maximum absorbance value (OD600) reaches1.316, butthat of wild-type E. coli was only0.393. Using the relative quantification2-△△Ctmethod of FQ-PCR to measure expression of PsTrx gene from Pseudoalteromonassp.ANT178, we found that the expression level of PsTrx at high salinity (5%-9%NaCl) was higher than low salinity (3.3%NaCl). And, the expression level at9% salinity was2.5times higher than that of control group (3.3%NaCl). This resultindicated that PsTrx gene played an important role in the mechanism of saltresistance in bacteria, which would provide a new gene for salt tolerance breeding.(3)TrxR gene from the strain Pseudoalteromonas sp.ANT178was cloned andnamed PsTrxR. It had a complete gene open reading frame, and its size was951bp.Using bioinformatics methods to analyse PsTrxR gene sequence, we found thatPsTrxR gene encoded316amino acids. The protein theoretical molecular weightwas33.7kDa, and its pI was4.98. We speculated that the key active sites wereCys136and Cys139, and its structure contained5α-helixs and18β-folds. ThePsTrxR gene was transfered into E.coli BL21to realize heterologous expression.TrxR of Pseudoalteromonas sp.ANT178was purified by Ni-affinity chromatogra-mphy and measured its enzyme activity. It is showed that its specific activity was29.71U/mg protein and yield reached35.75%. Its optimum temperature was20℃.It was inactivated for40min at50℃, and the enzyme activity decreased to66%.Using DTNB as substrate to measure enzyme kinetics parameters, we knew thatthe value of Kmand Vmaxwas1.15mM and5.17nmol/mL/min, respectively. It wascompletely inhibited by denaturant DTT, but little affected by metal ion Zn2+, andits relative activity was up to71.38%. |
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