| Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley because it reduces grain yield and quality and results in the contamination of grain with mycotoxins. Benzimidazole fungicides, including carbendazim (methyl benzimidazol-2-yl carbendazim, MBC) and thiophanate, have been relied to control the disease for thirty years. However, the appearance of resistance has hindered the continuous use of the fungicides, which causes severe prevalence of the disease. Because of the appearance of resistance, there is a current need to screen and develop new compound to control FHB. The mechanism of F. graminearum to MBC has been studied by genetic transformation and site-directed mutagenesis. The results have showed that site mutation on β2-tubulin could cause resistance of different levels, including F167Y, E198K, E198L and F200Y. There is no report of resistant mechanism on the protein level up yet now.Microtubules consist of repeating α/β tubulin heterodimers, and because of tubulin’s GTPase activity, they are highly dynamic. Microtubule dynamics can be inhibited by many antimitotic agents, and such inhibition is an important mode of action of anti-tumor drugs, antiprotozoal compounds and fungicides. Because of the success of tubulin-binding agents in anti-tumor drug screening, there is continuing interest in discovering new substances that interfere with microtubule-mediated processes. Though tubulins have been used as the target to screen and develop anti-tumor agents and anti-leishmanial drugs, fungal tubulins have not been used to screen for fungicides.In research concerning the development of an screening model for detection of anti-fungus compounds based on α/β tubulin assembly in vitro, and studying the resistant mechanism on protein level, the α-and β-tubulins of F. graminearum have been cloned on the pET vectors and expressed in Escherichia coli, and the polymerization in vitro and binding kinetics with carbendazim were also studied. The results are as follows.a-tubulin genes were amplified from F. graminearum cDNA and cloned to the vector pET30a+, then transformed into the host Rossatta (DE3) pLysS. After the positive clones were screened by the colony PCR and double digestion, the induced fusion proteins were obtained and verified by SDS-PAGE and Western blot. The positive clones which could express more fusion protein were screened, however, the fusion proteins formed mainly inclusion bodies. The molecular weight of fusion proteins were confirmed to be52.1kD and55.9kD by SDS-PAGE, which also showed specific activity to anti-6xHis monoclonal antibody. After washing of inclusion bodies using buffer containing2and3mol/L urea, the purity of fusion protein would increase. The soluble fusion protein was obtained by dialysis to binding buffer and then a-tubulins were purified by HisTrapTM HP Columns. The purified tubulins can be used in the studies of new fungicide screening in vitro that target tubulin.β2-tubulin of F. graminearum was expressed in E. coli in soluble form and purified by HisTrapTM HP Columns. β2-tubulin gene contained in the plasmid,(pET32a+-β2-tubulin) was amplified, cloned to the vector pET30a+and, then transformed into the hosts:Rossatta (DE3) pLysS and BL21(DE3). After the positive clones were screened by the colony PCR and double enzymatic digestion, the induced fusion proteins were obtained and verified by the SDS-PAGE and Western blot. In order to express the fusion protein in soluble form, the inducing factors, including temperature, induction time, IPTG (Isopropyl P-D-Thiogalactoside) concentration, cell density, medium composition and hosts, were screened. The positive clones which could express more fusion protein after induced were screened, however, the fusion proteins formed inclusion bodies. The molecular weight of fusion proteins were confirmed to be52kD by SDS-PAGE, which also showed specific activity to anti-6×His monoclonal antibody. After the optimization of imidazole concentration in binding and wash buffer, the soluble fusion protein was purified and its structural integrity was preserved through the purification process by the verification of western blot. The methods described here can be used to express and purify other recombinant proteins in soluble form in E. coli. The purified fusion tubulin can be used in the studies of tubulin-target drug resistant mechanisms as well as high throughout screening of new fungicide.The expressed a-tubulins existed in both soluble and insoluble forms, and the expression of soluble form was affected by isopropyl-β-D-thiogalactoside (IPTG) concentration, incubation temperature, incubation time, and culture density. The soluble proteins, which were purified by Ni2+affinity chromatography, were recognized by His-tag and a-tubulin antibody. The structural integrity of a-tubulins had been preserved throughout the expression and purification processes and the a-tubulins were suitable for high-throughout screening of candidate fungicides.After purified, dialyzed and concentrated, F. graminearum tubulins were polymerized. It was the first report that tubulins of F. graminearum polymerized in the absence of microtubule-associated proteins (MAPs). Polymerization was assayed in the PIPES buffer, where the concentration of tubulin and GTP was1mg-mL-1and2mmo1·L-1respectively. GTP can initiate the polymerization at37℃. Tubulin polymerization was assayed turbidimetrically at350nm for60minutes with an12seconds interval on96-well plates, using a Molecular Devices VersaMax microplate reader equipped with temperature controllers. There was no effect of DMSO (Dimethyl sulfoxide) on polymerization. The results showed that the assembly of of a2/β2-tubulin was higher than that of α1/β2-tubulin and the absorbance did not increase at350nm in the presence of carbendazim, which was consistent with sensitivity of the starting strain2021to this fungicide. Carbendazim had no effect on polymerized tubulins mixture and the absorbance did not decrease, however. There were different inhibition types of pesticides on the polymerization, which were not entirely consistent with their antifungal activity in vitro. The screening model based on the polymerization assay of F. graminearum α/β2tubulin can be used on the primary screening of new compound which targets tubulin.β2-tubulins originated from the resistant strains and site-directed mutants (SDMs)were cloned into pET32a and expressed in soluble form in E. coli after optimization of induction factors. The soluble β2-tubulins were purified and there were no differences of total fusion protein and soluble forms of amino acid substitution on β2-tubulin. The purified β2-tubulins could recognize the anti-His-tag and anti-β-tubulin monoclonal antibody, indicating that the structural integrity of β2-tubulins had been preserved throughout the expression and purification processes. Polymerization degree between α1-tubulin and β2-tubulins expressed by pET32a was consistent with that with P2-tubulin expressed by pET30a, so the unwanted amino acids on N terminals of P2-tubulins, including Thioredoxin, His-tag, S-tag, had no effect on polymerization. Mutation on β2-tubulin could promote assembly, where mutation on site198had the most dramatic ability. Carbendazim had no effect on polymerization in vitro between β2-tubulins of SDMs and a1-tubulin, implying that the binding force between carbendazim and β2-tubulins of SDMs was weaker than β2-tubulin of wild type strain which was sensitive to carbendazim.The in vitro binding characteristics of carbendazim with β-tubulins of different mutation sites, including Y50C, F167Y, E198K, E198L, F200Y and two mutation sites,(G17S+F167Y), were studied by fluorescence quenching. Fluorescence of all these recombinant tubulins was quenched following the addition of carbendazim, in which the β2-tubulin of wild sensitive strain had the highest quenching rate and the β2-tubulin with two mutation sites has the lowest, but the quenching rate of moderately resistant strains were higher than that of highly resistant ones. In the equilibrium binding studies, carbendazim with20μmol·L-1concentration could achieve the highest quenching rate for all recombinant tubulins, so the concentration was used in the binding kinetics experiment. The apparent association constant Ka)of β2-tubulin of site-directed mutants was negatively correlated with the resistant level of starting strain to carbendazim. However, the dissociation rates (Koff) was positively correlated with the resistant level. So that was affinity constant (Ka), which mean that the strain with high resistant level had the large Ka value. The Ka value of β1-tubulin to carbendazim was intermediate between β2-tubulin of wild sensitive strain and low resistance, implying that β1-tubulin contributed to the carbendazim resistance of F. graminearum. |
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