Expression And Functional Identification Of Glyphosate-resistant Gene From Cotton

Glyphosate is a widely used, broad-spectrum, inner-absorption conducting herbicide. Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase. EPSP synthase is the sixth enzyme of the shikimic acid pathway, which is essential for the biosynthesis of aromatic amino acids in algae, higher plants, bacteria and fungi. EPSP synthase catalyzes the conversion of shikimate-3- -phosphate (S3P) and phosphoenolphyruvate (PEP) to yield EPSP and inorganic phosphate (Pi). Glyphosate is a competitive inhibitor of PEP, as it occupies the binding site of PEP, mimicking an intermediate state of the ternary enzyme-substrates complex. Glyphosate has been world wide used to control weeds, because of the simply structure, low cost, high effective, lower deposition and accordance with the environment. Unfortunately toxicity to all plants makes it difficult to apply glyphosate to crops. In recent years, with the large-scale planting of glyphosate-resistant crops all over the world, applications of glyphosate become increasingly broader. Glyphosate-resistant crops have been commercialized from Monsanto Company. The study about glyphosate-resistant crops is lagging and hasn't obtained intellectual property rights in China. Consequently development of glyphosate- resistant crops is significant.The target gene used in this study was the glyphosate-resistant gene which was cloned from glyphosate-resistant cotton. The recombinant plasmid pET-HW199 had been transformed into E.coli BL21(DE3) and then induced by IPTG to obtain protein. The plant expression vector pBIHW199 had been transformed into LBA4404. Transgenic tobacco plants obtained via Agrobacterium-mediated leaf disc transformation and their sensitivity to glyphosate was tested.The target gene and the prokaryotic expression vector pET-32a(+) were digested with HindⅢ,SacⅠ,the purified objective gene was inserted into the compatible sites of prokaryotic expression vector pET-32a(+).The recombinant plasmid pET-HW199 had been transformed into E.coli BL21(DE3) and then induced by IPTG to obtain protein. Then the expression products were analyzed by SDS-PAGE. The result implied that the glyphosate-resistant gene was expressed at high level in 5h,37℃by SDS-PAGE analysis.The glyphosate-resistant gene and the plant expression vector pBI121 were digested with EcoRⅠ, the purified objective gene was inserted into the plant expression vector pBI121. The recombinant plasmid pBIHW199 had been transformed into LBA4404. Transgenic tobacco plants obtained via Agrobacterium-mediated leaf disc transformation and Kanamycin screening test. DNA extraction from transgenic tobacco plantlets, The PCR result showed that 13 plantlets in 54 plantlets display positive reaction, Southern blotting further confirmed the foreign gene has been transferred into tobacco plants. The herbicide glyphosate-resistant of tobacco has been identified by experiment. The vitro leaves of transgenic and non-transgenic tobacco plants were daubed with different concentrations of glyphosate. Results showed that the transgenic tobacco was tolerant to glyphosate up to 0.5% concentration, while the non-transgenic plants suffered serious at the concentration of 0.2%. The glyphosate resistance of tobacco plants results showed that the transgenic tobacco than the non-transgenic plants glyphosate-resistant has improved greatly.