Gene Cloning And Transgenic Plants Of MnSOD From The Thermophilic Fungi Thermomyces Lanuginosus And Thermoascus Aurantiacus Var.Levisporus

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 play a critical role in the defense against oxidative stress. Salt stress is the main factor that seriously affect on plant growth and development. 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.Thermomyces lanuginosus and Thermoascus aurantiacus var.levisporus are widely distributed soil-inhibiting fungi of considerable interest producers of thermostable enzymes, including glucoamylase, cellulase, endocellulase, xylanase, and laccase. The fungi have great value in research and application. In my lab, a MnSOD gene zjmnsod was isolated from Thermomyces lanuginosus. The full length of zjmnsod cDN A gene is791bp, which contained an ORF of654bp encoding217amino acids. The nucleotide sequence has been deposited in the GenBank database under accession number EU364837.The other MnSOD gene esjmnsod was isolated from Thermoascus aurantiacus var. levisporus, the gene contained an ORF of696bp encoding231amino acids. The MnSOD gene has been registered in Genbank with accession number EF428323.To investigate the function of zjmnsod and esjmnsod fromThermomyces lanuginosus and Thermoascus aurantiacus var.levisporusunder salt stress in plant, the zjmnsod and esjmnsod were subcloned into the expression vector pROK Ⅱ downstream of the35S-CaMV promoter, respectively. The constructs were first introduced into tobacco via Agrobacterium tumefaciens LBA4404and verified by PCR, RT-PCR. The results indicated that transgenic tobacco plants could enhance salt tolerance than the wild plants at the seed germination and seedlings growth stage.The SOD-transformed plants exhibited increased resistance to leaf infection with the fungi Alternaria alternata. Then, the zjmnsod and esjmnsod genes were subcloned into the expression vector pU1301downstream of the Maize Ubiquitin promoter, respectively. The constructs were first introduced into paddy rice via Agrobacterium tumefaciens EHA105.