Abstract: 【Objective】In the author’s previous studies, it was found that PcSOD3 is up-regulated after exposure to thermal stress, acaricide abamectin, heavy metal and UV-B ultraviolet irradiation, which suggested that PcSOD3 played an important role in the response of P. citri to various adverse environmental stress. In order to further elucidate the physiological functions of PcSOD3, the biochemical characteristics of its recombinant protein expressed in Escherichia coli was investigated. 【Method】The pET28a-PcSOD3 expression plasmid for the heterologous expression in E. coli was constructed. The recombinant protein was purified by using a Ni⁺ affinity chromatography column, and confirmed by the Western blot analysis. The enzymatic activity was measured using a SOD total activity assay kit. Moreover, the effects of various pH values of the reaction system and different temperatures for preincubation on the recombinant enzyme were tested using the same kit. Using the Kirby-Bauer disc diffusion assay method, the diameters of inhibition zones of the E. coli overexpressing PcSOD3 were measured by exposing to chromic chloride, t-butylhydroperoxide and cumene hydroperoxide. 【Result】 PcSOD3 was successfully expressed in the BL21 (DE3) strain of E. coli. The best condition for the induction and expression of the recombinant protein was obtained, under which cells were cultured at 18℃ and 160 r/min, IPTG was added to a final concentration of 0.4 mmol·L^-1, the inducing time continued for 18 h. The resulted PcSOD3 recombinant protein was further confirmed by the Western blot analysis, with the molecular weight of 25.3 kD. Moreover, the PcSOD3 recombinant protein was active in the WST-1 activity assay system. Based on the assay system, it was found that PcSOD3 recombinant protein was mostly active (activity was about 47.3 U/mg protein) when the pH of the reaction system was 7.0, while it was mostly active (activity was about 40.2 U/mg protein) when the preincubation temperature was 25℃. As indicated by the Kirby-Bauer test, the diameters of inhibition zones of the E. coli overexpressing PcSOD3 which were exposed to chromic chloride (CdCl₂), were about 20% smaller than the control. Similarly, overexpression of PcSOD3 in E. coli had significantly reduced the inhibition zone (around 25%) caused by exposure to t-butylhydroperoxide at the concentration of 150 mmol·L^-1, compared to the control. Meanwhile, E. coli expressing PcSOD3 showed 15% reduced inhibition when exposed to cumene hydroperoxide.【Conclusion】PcSOD3 was functionally expressed in E. coli. Purified PcSOD3 recombinant protein was obtained. Furthermore, the biochemical characteristics of the recombinant enzyme such as the optimal pH and preincubation temperature was determined. The recombinant enzyme showed a significant antioxidation activity in vitro by WST-1 assay method. The results of Kirby-Bauer disc diffusion assays showed that the E. coli overexpressing PcSOD3 had a significantly elevated tolerance to the oxidative stress, which has proven that PcSOD3 has the antioxidant function. Results of the experiment further revealed that PcSOD3 played a crucial role in the tolerance of P. citri to oxidative stress.

Key words: Panonychus citri, superoxide dismutase (SOD), biochemical characteristics, recombinant protein, Kirby-Bauer test