PMID: 18720837Aug 30, 2008Paper

Isolation, prokaryotic expression and activity analysis of thymidylate kinase (tmk) gene from Phytoplasma of wheat blue dwarf

Wei sheng wu xue bao = Acta microbiologica Sinica
Bei LiXing'an Hao

Abstract

Wheat blue dwarf (WBD) is an important disease in winter wheat district, which causes serious losses in wheat production. Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to dTDP in the de novo and salvage pathways of dTTP synthesis in both prokaryotes and eukaryotes. In order effectively control this phytoplasma, we isolated the thymidylate kinase gene of WBD phytoplasma, and analyzed the catalytic activity of TMK protein. tmk gene was amplified from the phytoplasma of WBD, the amplicons were digested with EcoR I and Hind III and then inserted into expression vector pET-30a(+). The polyHis-tagged TMK was expressed in E. coli BL21 (DE3) and fusion protein was obtained and purified by Ni-NTA column. The TMK activities were measured by the method of en-zyme-coupled assay involving Mg2+, dTMP and ATP. Two genes, tmk-1 and tmk-2 were obtained, with the molecular weight of 630 bp and 624 bp. Both of them encoded an amino acid sequence with three conserved functional motifs which related with binding NTP/NMP. The fusion protein, TMK-2 had a higher catalytic activity (112.41 U/mg) than TMK-1 (16.4 U/mg), and its optimum catalytic conditions were 32 degrees C, pH7.3, 1.5 mmol/L Mg2+ and 1 mmol/L ATP. TMK-1 and TMK-2 had c...Continue Reading

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