The Effect Of Cordyceps Militaris Mn-SOD Transgene On Tobaccos Resistance To Virus Infection And Its Expression In Prokaryotic Cells

Transgenic tobaccos (Nicotiana tabacum cv. YunyanS5) seedlings containing the Mn-SOD gene from Cordyceps militaris were regenerated on kanamycin selective MS medium. After screening by PCR, Southern-blotting and Western blotting, the homozygous transgenic lines of tobacco were obtained, among which the line 14-18-1 was chosen for inoculation with purified Tobacco mosaic virus (TMV) in order to clarify the role of Mn-SOD in the compatible interaction between the virus and transgenic tobaccos. The results showed that the transgenic tobaccos expressing Mn-SOD were more susceptible to TMV infection, compared with non-transgenic tobaccos. There were significant differences in the virus accumulation, Mn-SOD expression and MDA levels between the transgenic plants and the non-transgene controls after TMV inoculation, suggesting that during the process of virus infection, non-transgenic tobaccos were able to keep higher active oxygen level than that of the transgenic tobaccos. These differences may result in some extent of defence response to virus infection through reducing the expression level of Mn-SOD in the wild type plants, whereas the active oxygen was decreased to a certain level that facilitate TMV infection due to over-expression of heterogenous Mn-SOD in transgenic plants.A prokaryotic vector containing the Cordyceps militaris Mn-SOD gene was constructed for expression in E.coli BL21 (DE3) strain by IPTG induction. Based on a series of experiments, the optimum method was developed for over-expression of the protein in mini-culturing with flaskes. With the protocols of ultrasonication, affinity-chromatography, gel filtration, a simple highly effective method has been established for preparation of the Mn-SOD protein, by which the purity was up to 98% in a recovery ratio of 65%. In addition, thermal stability of the purified Mn-SOD was also characterized. This research can be used for the further research and possible application of the Mn-SOD protein from Cordyceps militaris.