Cloning And Expression Of Superoxide Dismutase Genes From The Thermophilic Fungus Chaetomium Thermophilum And The Salt Tolerance Of Transgenic Tobaccos

The superoxide dismutases (SOD, EC1.15.1.1) are metallo-enzymes that catalyze the dismutation of superoxide(O2.^-) to hydrogen peroxide(H₂O₂) and molecular oxygen(O2). They have play a critical role in the defense against oxidative stress. There are three general classes of SODs in organisms, which differ in their metal cofactors: copper zinc-containing SOD (Cu,Zn-SOD), manganese-containing SOD (Mn-SOD)and iron-containing SOD (Fe-SOD).In recent years, the important role of SOD in food, pharmaceutical, cosmeceutical industries and agriculture has been demonstrated. The SOD used in the market mainly from room temperature organisms. However, SOD is easily degraded at high temperatures and it can not be stored for long periods. Due to high production costs, poor thermal stability, low activity and yield, the application of SOD is restricted in agriculture and industrial applications. Therefore, there has been increasing interest in research and development of thermostable SODs from thermophilic organisms.Chaetomium.thermophilum is a widely distributed soil-inhibiting fungi of considerable interest producers of thermostable enzymes, including glucoamylase, cellulase, endocellulase, xylanase, and laccase. The fungi has great value in research and application.In this study, a Mn-SOD gene was isolated firstly from C.thermophilum CT2 by RT-PCR and Tail-PCR. Sequence analysis showed that the full cDNA of the gene was 684bp, encoding 227 amino acids. The gene was highly expressed in Paster.pastoris and the recombinant protein was purified by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow anion exchange. SDS-PAGE showed that the recombinant protein was about 23kDa, similar to the deduced molecular weight. The optimal temperature and pH were 60℃and 6.0, separately. The enzyme was relatively thermostable, after incubated at 70℃for 60 min still remained 71% activity.In this study, a Cu,Zn-SOD gene from C.thermophilum was also cloned and highly expressed in Paster.pastoris, Six days after induction, this strain had the highest SOD activity of 1,660 U/ml culture, and its expression level was 1.54 mg/ml. The recombinant protein was purified by ammonium sulfate precipitation and DEAE-Sepharose Fast Flow anion exchange. SDS-PAGE showed that the recombinant protein was about 17kDa. The recombinant Cu,Zn-SOD exhibited optimum activity at pH 6.5 and 60℃. The enzyme retained 65% of the maximum activity at 70℃for 60min and the half-life was 22min and 7min at 80 and 90℃, respectively. The recombinant yeast GS115 (harboring pPIC9K/mnsod or pPIC9K/czsod) and control GS115 (harboring empty pPIC9K) were treated with different concentration of salt and oxidation stresses such as paraquat, mandione and H₂O₂. The results revealed that the recombinant yeast cells have a higher stress resistance than control cells. It also proved that the mnsod and czsod genes from C.thermophilum have the ability of salt and oxidation resistance.Salt stress is the main factor that seriously affect on plant growth and development. Therefore, there is interest in breeding transgenic plants that are tolerant of saline soil. Salt-stress adaptation mechanisms are being intensively studied in model organisms. The genetic engineering is an effective way to improve plant salt stress resistance. One of the basis for plant genetic engineering is study on the function of stress- related gene. Exposure of plants to environmental stresses, including salinity, drought, cold and high temperatures, can lead to the generation of reactive oxygen species (ROS). ROS can attack cellular macromolecules, generate lesions in DNA, cause membrane damage, and affect protein synthesis and stability, therefore it is important for plants to have effective ROS scavenging mechanisms.Superoxide dismutase activity is closely associated with defense ability of plants under the condition of salt stress. Physiological correlations between elevated SOD activity and stress tolerance have been reported, suggesting that the upregulation of SOD levels may enhance the stress-defense potential of plants. Various transgenic plants that express increased amounts of SODs have been generated for enhanced tolerance to environmental stress.To investigate the function of mnsod and czsod from C.thermophilum under salt stress in plant,the full-length mnsod and czsod cDNAs were subcloned into the expression vector pROKâ…¡downstream of the 35S-CaMV promoter, respectively. The constructs were first introduced into tobacco via Agrobacterium tumefaciens LBA4404 by the freezing transformation method and verified by PCR, Southern blot and RT-PCR. It was indicated that the mnsod and czsod gene had been recombined into tobacco genome, respectively, and transgenic tobacco plants were obtained.On the medium containing the different concentration of NaCl, expression of mnsod or czsod gene in tobacco can showed the higher percentage of seed germination. Both of the transgenic lines exhibited increased root growth compare to WT plants.These results indicated that transgenic tobacco plants could enhance salt tolerance than the wild plants at the seed germination and seedlings growth stage.Salt stress resulted in membrane injury and caused MDA content and the extent of the membrance lipid perioxidation rising in plants. In this study, salt stress increased MDA content in all Plants. However,the values of transgenic plants measured were much lower than those of wild-type, which indicated that the extent of the membrance lipid perioxidation in transgenic tobacco was lighter than that in wild type tobacco. These results indicated that the transgenic plants showed enhanced salt tolerance compared with wild-type under salt stress conditions.Under salt stress, the activity of SOD increased first and then decreased in both wild types and transgenic tobacco plants. Furthermore, the transgenic tobacco plants always sustained higher SOD activity than wild-type plants.The study showed that the SOD-transformed plants exhibited increased resistance to leaf infection with the fungi Colletotrichum nicotianae and Alternaria alternate.The enhanced tolerance of transgenic lines to salt stress suggests that the mnsod and czsod genes from C.thermophilum are benefit for genetic engineering to improve plant tolerance to salt stress.