Obtainment And Glyphosate Tolerance Analysis Of MAM79 Transgenic Maize

AM79 aro A gene which encoding a bacterial 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) can be a good candidate gene for the development of glyphosate-tolerant transgenic crops. In our previous study, the redesigned and synthesized AM79 gene was named m AM79. In this study, the molecular identification and glyphosate-tolerant analysis of m AM79 transgenic maize obtained from early stage were conducted. Meanwhile, to generate more transgenic events with different insertional sites, the expression cassette of m AM79 was cloned into Ds transposable element to construct plasmid p3301-Zm Ds-Ubi Sp AM79, which was transformed into maize immature embryos using Agrobacterium-mediated transformation method. The m AM79 transgenic maize lines were obtained and their glyphosate tolerance was analyzed preliminarily. The main results are as follows:The PCR-based detection of m AM79 transgenic plants obtained from previous work showed that m AM79 gene was integrated into maize genome DNA. High expression of m AM79 gene in seven independent transgenic lines was detected via RT-PCR method. However, Western blot analysis indicated that there was a big difference among the seven independent transgenic lines, among which transgenic lines AM85 and AM72 had high levels of AM79 EPSPS proteins. When sprayed with glyphosate in the field, the two transgenic lines had the tolerance of four folds of commercial usage of glyphosate, whereas the other transgenic lines were significantly affected and all the non-transgenic plants were killed. The conclusion from this study confirmed that m AM79 gene could be used as an effective candidate gene to develop glyphosate-tolerant transgenic maize, and the obtained transgenic maize events could be used for the breeding of glyphosate-tolerant maize.The expression cassette of m AM79 was located in the Ds element to construct the plasmid p3301-Zm Ds-Ubi Sp AM79 which was transformed into maize and five Ds-m AM79 transgenic maize lines were obtained via Agrobacterium-mediated transformation method. The PCR analysis of T1 generation transgenic maize showed that they had the integration of the Ds-m AM79. RT-PCR results manifested that m AM79 gene was highly transcribed in five transgenic lines. When sprayed with glyphosate, it was observed that the transgenic plants had a better performance in growth and survival, compared with the non-transgenic maize. In this study, we got some Ds-m AM79 transgenic maize lines which showed good glyphosate tolerance, and the further work is to obtain more independent glyphosate-tolerant maize events with different insertional sites by crossing with active Ac transponson lines.