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


METHODS
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.


RESULTS
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.


CONCLUSIONS
TMK-1 and TMK-2 had conserved functional motifs in their primary sequence, and suggested that they may function as TMK enzymes. But, the TMK-1-polyHis fusion protein had very low catalytic activity, the possible reason was that two highly conserved regions were absent in TMK-1, and it might function as another type of kinase in WBD phytoplasma. This experiment lay a foundation for further study of the TMK function in infection and reproduction of WBD phytoplasma.
Li
Bei
B
Northwest Agriculture and Forestry University, Shaanxi Key Laboratory for Agriculture Molecular Biotechnology, Yangling 712100. zero0707007@163.com
Ji
Lingling
L
Wu
Yunfeng
Y
Hao
Xing'an
X
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D055162:Biocatalysis; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D045582:Phytoplasma / Q000235:genetics*; D010935:Plant Diseases / Q000382:microbiology*; D011387:Prokaryotic Cells / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature; D014908:Triticum / Q000382:microbiology*
D000255:Adenosine Triphosphate; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase; D008274:Magnesium