Agrobacterium-mediated Gene Resistant Glyphosate Maize18-599Genetic Transformation Of Immature Embryos

Maize(Zea mays subsp. Mays L), as one of the three major grain crops, not only plays an important role in food security, but also provides a good raw material for the bio-energy manufacture. With the population growth, the people’s living atandard improvement, and the animal husbandry development, there is a significant increase in the demand for corn.Weeds reduce yields by competing with maize for sunlight and fertilizer, which seriously restricts the maize production. Artificial weeding and mechanical weeding are both time-consuming, laborious and no good effect; chemical weeding is has been become an important part of modern agricultural technology because it is convenient, economical and effective. Currently, the largest and widely using herbicide is glyphosate in the global agricultural production. Glyphosate herbicide was limited to weed in maize production because of broad-spectrum weed control and lethal effect for maize. Therefore, to develop new glyphosate-resistant maize varieties with independent intellectual property rights, and to test, demonstrate and promote them, can reduce weed control costs and labor intensity, improve labor efficiency, and enhance the international competitiveness of Chinese maize products.In this study, our main objective is to transfer glyphosate resistance gene2mg2-epsps into maize inbred line18-599R immature embryos using Agrobacterium tumefaciens. The main research contents are as follow:to explore the effect of heat shock and centrifugation for immature embryos; to optimizate transgenic condition, such as the size of immature embryo, the selection concentration, the cephalosporins concentration and so on; and to establish good Agrobacterium tumefaciens-maize immature embryos transgenic system; and to obtain glyphosate resistance transgenic corn plants. Transgenic plants were analyzed by PCR, and PCR products were sequenced. PCR and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect and identify T1plants, and the results indicated that the2mg2-epsps gene was integrated into the maize genome. Positive plants were tested using glyphosate, and the results validly proved that the transgenic offspring had glyphosate resistance. The main results are as follows:1To acquire target gene. The2mg2-epsps gene was amplified from a expression vector P35S-2301-Epsps using High-fidelity enzyme, and was sequenced. Using the NCBI Blast and ORF Finder analysis, the results showed that the length of cloned sequence was1563bp encoding464amino acid, and99%and100%homology with the2mg2-epsps gene in nucleotide and amino acid, respectively.2Construction of expression vector. The pMD19T-2mg2-epsps and pCAMBIA3301empty vector were both digested by two restriction enzymes. Then target fragments were recycled, ligated, and transformed. The results from restriction enzyme digestion and sequencing showed that the target gene had no mutations during the process of construction of expression vectors.3Optimization system for genetic transformation of maize immature embryo. The factors that effected Agrobacterium infecting maize immature embryo were discussed, such as Pre-treatment of the maize18-599R isolated immature embryo by heat shock and centrifugation, the size of immature embryos, the concentration of Cefotaxime, the concentrations of glyphosate, and so on. The results showed that heat shock at40℃with3min, cold treatment at25℃with2min, and no centrifugation were the most suitable pre-treatment conditions. And under the conditions, the ratio of obtaining resistant callus is the highest (52%).400mg/L of cephalosporin could not only inhibit Agrobacterium growing and also maintain maize immature growing well. Among different concentrations of glyphosate screening infected immature embryos screening concentration was ideal for1.5-2.0mmol/L,4PCR identification of transgenic plants. Of96To plants,15transgenic plants are positive by duplicate PCR. The result showed that the exogenous gene had been integrated into the genome of maize. T1transgenic seedlings were tested by PCR, which showed that the4lines were positive. Those positive plants were detected by RT-PCR, which indicated that the glyphosate-resistant gene transcription at the mRNA level has been effectively. Those positive plants were screened by glyphosate, and the result showed they had glyphosate resistance.