Isolation, Prokaryotic Expression And Activity Analysis Of Thymidylate Kinase Gene (tmk) From Phytoplasma Of Weat Blue Dwarf

Wheat blue dwarf (WBD) is the first phytoplasma disease of wheat inated by Psammotettix striatus L. in China. It is the main epidemic disease in arid and semiarid areas in northwest of China, and it can cause a great loss in the production process of winter wheat. Therefore, the study of WBD is an important problem for a long time. Thymidylate kinase (TMK, EC 2.7.4.9), catalyses the transfer of phosphoryl group from ATP to dTMP, and make the phosphorylation of dTMP to form dTDP in the presence of magnesium ion. Therefore, it is an important enzyme in the salvage pathways of thymidine deoxyribonucleotides in phytoplasma, and it is crucial to DNA synthesis and repair.There were some difficulty of culturing phytoplasmas in vitro and the content of phytoplasma in plants is too low which restricted the study. In this study, the tmk gene of wheat blue dwarf phytoplasma was isolated, prokaryotic expressed, and the TMK protein activity was assayed in vitro. The results are as follows:(1) Isolation of thymidylate kinase gene (tmk) from phytoplasma of Wheat Blue Dwarf: Two amplified fragments named tmk-1 and tmk-2, 630 bp and 624 bp respectively, were obtained by PCR from total DNA of WBD phytoplasma samples which were collected from hancheng city in Shaanxi province. The two genes encoded proteins of 209 and 207 amino acids, respectively, GC % of which were 29.68 % and 28.37 %. Comparing with other phytoplasmas, homologic analysis showed that the nucleoside sequence and the deduced amino acid sequence of the protein encoded by tmk-1 of WBD phytoplasma shared over 94 % identity with Clover phyllody (CPh) and tmk-a of Onion Yellows phytoplasma (OY), and tmk-2 also shared over 94 % identity with Aster yellows witches'-broom (AYWB) phytoplasma and tmk-b of OY . Those revealed that WBD phytoplasma was closely related to the phytoplasmas of 16Srâ… g roup. The sequences of tmk-1 and tmk-2 gene were submitted to the GenBank, the numbers were EU183350 and EU183351, respectivly.(2) Structure characteristics of thymidylate kinase: The analysis of primary sequences of proteins encoded by tmk genes (TMKs) of WBD phytoplasma showed that, WBD-TMKs had three conserved functional motifs involved in NTP/NMP binding, which were P-loop domain, TMP-binding motif and LID domain. The facts that TMK-1 and TMK-2 have the same conserved functional motifs in the primary sequence indicated that they may function as TMK enzymes. But in the sequence of TMK-1, a residue W was in place of Y in the DRY tripeptide in TMP binding motif which were essential for catalytic activity, and furthermore, two highly conserved regions, N′- TKEPGG -C′, downstream of the P-loop motif, and N′- PAL -C′, upstream of the TMP binding motif, were absent, both above mayeffect impair the catalytic activity of TMK-1.(3) Prokaryotic expression of tmk gene: Constructed plasmid pET30-tmk and pBV221-tmk were transformed into E.coli. respectily, and then induced in different conditions. The results of SDS-PAGE showed that both tmk-1 and tmk-2 gene were expressed effectively in E.coli.. But the expressed fusion proteins of pBV221- tmk were inclusion bodies, so pET30a was selected as the prokaryotic expression vector. There is maybe some toxin from antigenic membrane protein. Then Constructed plasmid pPIC9K-AMP, and test the sequence with PCR,enzyme and sequencing. Although the expression of Amp was not successful, the results provide another evidence on diversity of phytoplasmas. The optimization of induced temperature and time revealed that, the mass of soluble proteins induced 14h under 16℃conditions can meet the requirements of following tests. The target fusion proteins were purified by Ni-NTA column, and eluted with buffer containing 500mM imidazole.(4) Acivity analysis of TMK enzymes: The results showed that, the fusion protein, TMK-2 had a higher catalytic activity (112.41 U/mg) than TMK-1 (1.64 U/mg), and its optimum catalytic conditions were 32℃, pH7.3, 1.5 mmol/L Mg²⁺ and 1 mmol/L ATP. When the His tag was resected by thrombin, the activity of TMK-2 and TMK-1 didn't changed significantly, this fact revealed that the His tag on the N-terminus of fusion protein did not effect the catalytic activity of TMK-2, and inhibit that of TMK-1. There might be several possible reasons why TMK-1 had low catalytic activity. The optimal reaction conditions for TMK-1 may differ from those of TMK-2. The fused poly-His tag on N terms of the fusion protein may inhibite the catalytic reactions in the experiment. In addition, TMK-1 may function as another type of kinase in WBD phytoplasma. Finally, two highly conserved regions were absent in TMK-1, may impair the catalytic activity of TMK-1.