Mutation Of EPSP Synthase Gene Of Halomonas Varabilis And Breeding Of Maize Inbred-lines With Resistance To GlyPhosate

Glyphosate is an exceptionally reliable, phloem mobile, broad-spectrum herbicide with less soil residual activity. Glyphosate is the most widely used herbicid in agriculture.It has the largest production volume and most commercial value in the world. Glyphosate is a competitive inhibitor of EPSPS (5-enolpyruvyl-shikimate-3-phosphate synthase), which is essential for the shikimate pathway. The aromatic acids synthesis can be interrupted when plants were treated with glyphosate. Because of the shikimate pathway serves to biosynthesize the aromatic acids and other secondary metabolites for the survial of plant and microorganism, it’s essential in microorganism and plant. Since glyphosate is non-selective, while it kills weeds in field, crops also been damaged. Therefore, molecular breeding for glyphosate-resistant crops is an important means of weed control. Transgenic crops with high dose glyphosate tolerance could be produced by introducing the glyphosate resistant EPSPS genes into crops.Maize (Zea mays L) as the world’s largest crop of foods, have a big impact on feed, energy and industry, has been widely planted in the world. With the continuous generation of resistant weeds, the negative effects of weeds on mazie production have been increasingly growing. It is great significance to breed glyphosate -resistant maize varieties with improved yields in agricultural production.In order to increase the glyphosate resistance of the GM maize, cDNA of the EPSPS gene cloned from Halomonas varabilis (referred to as H. Var-EPSPS) was modified by maize codon bias optimization. In this study, we used Splicing Overlapping Extension PCR to site-directed mutagenesis of the template sequence ofEPSPS based on designs and produce two genes of EPSPSM1 and EPSPSM2. In the sequence of EPSPSMl, the 295th base is T instead of G and the coding amino acid at 99th changes from Ser into Ala, and the 299th base is G instead of C and the coding amino acid at 100th changes from Gly into Ala. In the sequence of EPSPSM2, except the mutations in EPSPSM1, the 292th base is A instead of T and the coding amino acid at 98th changes from Asn into Tyr. Three-dimensional conformation prediction indicated that the conformation at the substrate binding site of EPSPSM1 and EPSPSM2 have occurred some changes.To determine the effects of the EPSPSM1 and EPSPSM2 on transgene maize plants for the resistance to glyphosate compared with the EPSPS gene, they were recombined into the plant expression vector to transfomate maize cells by Agrobacterium-mediated transformation of shoot tips respectively. The EPSPSM1 or EPSPSM2 gene is initiated by the Ubiquitinl promoter from maize and is terminated its transcription by the 3’UTR of GluB5 gene from rice. And the modified bar gene that initiated by the constitutive promoter, an enhanced CaMV35S promoter, and has a terminator, the 3’UTR of GluB5 gene from rice, is also recombinated into the same plant expression vector. The bar gene also be modifyied by maize codon bias optimization. The plant expression vector with the gene of interest was introduced Agrobacterium LBA4404 to transform maize inbred lines Chang 7-2. The transgenic maize plants with dual herbicide resistance were produced.Molecular evidence of genetically modified maizeThe PCR results show that EPSPS M1 or EPSPS M 2 gene and the bar gene had been integrated into the maize. Among the EPSPS M1 maize T1 lines of 76 glyphosate resistance lines, positive results were detected in 32 lines, so the transgenic rate of TO plants was 42.1%. Among the EPSPS M2 maize Tl lines of 70 glyphosate resistance lines,29 of the lines showed PCR positive, the transgenic rate of TO plants was 41.4%. The PCR results of T3 lines from those PCR positive T2 plant were also positive. The Western Blot analyze demonstrated that the optimized EPSPS expressed in the T3 GM maize.Herbicide resistance of genetically modified maizeIn this study we compared the glyphosate tolerance of the GM mazie obtained in our lab. The GM maize plants contains H. Var-EPSPS, modified H. Var-EPSPS by maize codon bias optimization, EPSPS M1 or EPSPS M2, respectively. When the plants grew at the 4th leaf-stage, they were treated with 2.52 kg ae ha-1 glyphosate, which was 3 times of the recommended dosage to extirpate weed in the fields. The GM mazie plants with EPSPS MI or EPSPS M2 obtained in this study maintained good growth and showed an excellentglyphosate tolerance, and better than the GM maize with H. Var-EPSPS and modified H. Var-EPSPS by maize codon biasoptimization. The 2 GM maize in this study also have glufosinate tolerance, they can resistant to 0.412kg ae ha-1 glufosinate treatment in the third leaf-stage. In maize weed prevention,GM maize with a dual herbicide tolerance may be selectively intermittent spraying one herbicide or a herbicide mixture, so that we can not only better control weeds, but also delayed the evolution of resistant weeds.In summary,the GM maize with a dual herbicide resistance obtained in this study has an important significance in weeds control and lay the foundation for the growth of Chinese herbicide resistant maize.