Directed Evolution Of Cu/Zn-superoxide Dismutase And Use As A Safe Selectable Marker In Plant Transformation

Biosafety issues of selectable marker genes are the major choke points which restrict the process of genetically modified plants industrialization. The development of bio-security marker genes to substitute for antibiotics and herbicides resistance genes is viewed as an important and alternative strategy to solve the biosafety problem and promote the application of transgenic plants. In this study, Kluyveromyces lactis Cu/Zn-SOD was modified by in vitro directed molecular evolution to obtain high activity mutant gene which was used as a selectable marker gene with paraquat as selective agent. The feasibility of this new efficient screening syetem was systematically discussed.We constructed YEplac195-PSGA plasmid by cloning Kluyveromyces lactis Cu/Zn-SOD gene fused to GFP gene under PGK1 promoter. The plasmid was transformed into yeast W303 strain. The expression of Kluyveromyces lactis Cu/Zn-SOD gene in yeast was confirmed by colony PCR, fluorescence microscopic observation and fermentation added with paraquat.By the one-step gene replacement, sod1Δyeast mutant LN303 was constructed. Under high oxygen pressure produced by 1 mmol/L of paraquat added to CM-Ura medium, sod1Δyeast mutant was first used as an expression host. Hence, Cu/Zn-SOD efficient screening was established.For optimization of error-prone PCR system, the Mn2+ concentration was set of 0.5 mmol/L. By error-prone PCR, mutant library was established and screened,obtaining three mutations S1, S2, S3 (S99R/A92G, S135P, D126N) with increased enzyme activity, which were 2.6-, 1.5- and 1.2-folds compared to wild type, respectively. We used computer simulation of three-dimensional structure to primarily analyse structural changes and the possible molecular mechanism of increased activity of the mutant enzymes caused by the amino acid substitution. The result showed that, local electrostatic environment, internal hydrogen bond interaction, and the size and shape of activity channel greatly impacted on the enzyme activity enhancement.The plant expression vectors pBI121-Chl-SOD and pBI121-Chl-mSOD were constructed by fusion of Kluyveromyces lactis Cu/Zn-SOD gene, the mutant S1 (mSOD) gene to the chloroplast transit sequence from Arabidopsis thaliana glutathione reductase (Chl), respectively. Via Agrobacterium-mediated transformation of tobacco, 35 and 46 transgenic lines were successfully regenerated, respectively. PCR, RT-PCR, and chloroplast SOD activity assay demonstrated that the target gene was integrated into the tobacco genome, normally expressed and targeted into the chloroplast. The SOD activities of transgenic Chl-SOD plants and transgenic Chl-mSOD plants were respectively 1.8-and 5.3-folds of wild-type plants. Transgenic plants treated with paraquat and 200 mmol/L NaCl were investigated for apparent injury, membrane permeability, chlorophyll a fluorescence and antioxidant enzyme activities. Results showed that the transgenic lines showed different degrees of increased resistance to oxidative stress and salt stress, and the order of stress tolerance enhanced was: transgenic Chl-SOD plants>transgenic Chl-mSOD plants>wild-type plants. Therefore, over-expression of Kluyveromyces lactis Cu/Zn-SOD and mutant S1 (mSOD) gene in tobacco chloroplasts can enhance oxidative stress tolerance and salt stress tolerance of transgenic plants.A new security screening system was established based on Kluyveromyces lactis Cu/Zn-SOD mutant S1 gene as a selectable marker gene and paraquat as the selective agent. The optimal concentration of paraquat concentration was determined to be 6 ?mol/L. 96 normal phenotype of T0 independent transgenic tobacco events were produced via Agrobacterium mediated procedure using mSOD gene as a selectable marker gene and 6 ?mol/L paraquat selection. PCR, Southern blot and RT-PCR detection in transgenic plants demonstrated that mSOD gene was integrated into the tobacco genome and expressed. The highest transformation frequency was 39.26%. Compared with traditional screening system using the standard selectable marker gene nptⅡand a selection regime of 100 mg/L kanamycin, the ratios of mSOD-positive transgenic plants were respectively 2.16-and 2.1-folds for the transformation of pBI121-Chl-mSOD and pBI121’ plants. Genetic stability analysis of transgenic tobacco demonstrated most T0 transgenic plants contained single-copy insertion, with 3:1 segregation consistent with Mendel’s law.The germination rates of T1 seeds, height, and leaf number were recorded to inspect paraquat-induced oxidative stress and salt stress tolerance of T0 and T1 generation of transgenic plants. The result showed that both of them were higher than that of wild-type plant. This new security screening system avoided the use of antibiotic or herbicide resistance genes therefor relatively safe to environment and human. It offered other benefits, for example transformation efficiency improved and the plants growing period shortened. Selectable marker gene can also be used as target gene, which confer oxidative stress tolerance and salt stress tolerance of transgenic plants.