Thioredoxin(TRX-h)is a small molecular protein that catalyzes the reduction of disulfide bonds and plays an important role in regulating the response to oxidative stress.In order to study the structure and function of TRX-h,wild type Arabidopsis thaliana Columbia(Col-0)was used as the research material.The coding sequence(CDS)of TRX-h5 gene was obtained by reverse transcription-polymerase chain reaction(RT-PCR).The prokaryotic expression vectors of pET28a(+)-TRX-h5 and pET28a(+)-TRX-h5M(mutation at cysteine sites 39 and 42)were constructed in vitro and trans-formed into E.coli competent cell BL21(DE3).After induction,the fusion protein with six His tags was purified by nickel column.SDS-PAGE detected the target band at 14 kDa,which was consistent with the theoretical prediction.Bioinformatics analysis showed that TRX-h5 gene encoded 118 amino acids,the relative molecular weight was 13 122.32,the theoretical isoelectric point was 5.19,and the instability parameter of the protein was 26.74,which indicated that the protein was a stable protein.Point mutation analysis showed that the cysteine sites at 39 and 42 of TRX-h5 determined the catalytic activity of the protein,which provided a new idea for further exploring the protein function of TRX-h5 and the redox system of higher plants.
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