Research On The Novel Glyphosate-resistant Corn And The Selectively Terminable Strategy

The genetically modified(GM) corn was one of the initial launched and marketed crops in the history of agriculture, is also the most widely planted GM grain crops in the world. In recent years, the major categories of transgenic corn traits commercially available is glyphosate tolerance. Actively developing a new generation of glyphosate-resistant corn varieties with patents, is the only way for us to compete with foreign competition and challenges. There are three basic strategies that have been evaluated in order to introduce glyphosate resistance into crop species:over-expression of the sensitive target enzyme, detoxification of the glyphosate molecule and expression of an insensitive form of the target enzyme. In this study, an insensitive EPSPS gene was transfered into corn to achive the best event.However, lingering concerns about the potential health and environmental effects of genetically modified organisms have limited the acceptance of such seed lines and food products. In spite of the strict policy in transgenic regulation, the contamination and unintended spreading still occurred from time to time based on control methods of physical separation and isolation.So it is very important to find new strategies to prevent the potential hazards of transgene spreading. In this study, we report a built-in selectively terminable strategy to mitigate the possibility of transgene spreading into conventional or organic corn crops.The results are as follows:(1) The EPSPS gene G10was isolated from Deinococcus radiodurans R1(GI:8469109) in our laboratory previously. Phylogenetic analysis revealed that this EPSPS belongs neither to Class Ⅰ nor to Class Ⅱ EPSPS enzymes. The G10DNA sequence was synthesized to be optimized for expression in corn. In order to direct the EPSPS to the chloroplast, a chloroplast transit peptide from the acetohydroxyacid synthase of Zea mays was added in the5’end of the G10gene. The T-DNA plasmid p1300-pZmUbi-G10-p35S-actintron-G10containing two G10genes under the control of a corn ubiquitin promoter and a cauliflower mosaic virus35S promoter, separately. It was transformed into corn via a standard Agrobacterium tumefaciens-mediated method. In our transformation, total of208 independent transgenic events were obtained. The transgenic plants were identified by PCR、 Southern blot、Western blot、Tail-PCR and glyphosate spraying test. The results suggest that the transgenic event Anti-G-162is the best one. Furthermore, there was no significant phenotype difference observed between the T2plants and the control plants in field test. And it at least has the same glyphosate-resistant level as the Monsanto’s products in Zhe jiang Provience. Therefore, this study will provides research materials for the breeding of transgenic corn and also could be used to develop new transgenic corn hybrids.(2) As corn is a wind pollinated crop, the eventual introgression of transgene from transgenic hybrids into conventional or organic corn crops is likely if they are grown closely together. To avoid the potential hazards caused by transgene spreading and contamination, new technologies should be explored in addition to physical separation and isolation measures. Previously, we described a built-in strategy for containment of transgenic rice. In this study, we report a method to create glyphosate tolerant transgenic corn events that are sensitive to nicosulfuron. This was achieved by constructing a T-DNA plasmid with the glyphosate tolerant5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene and an RNAi cassette suppressing the expression of the nicosulfuron detoxifying enzyme gene CYP81A9. Our experiments showed that the transgenic corn event R450-58with the built-in selectively terminable strategy was sensitive to nicosulfuron and could be killed by spray of nicosulfuron at the regular dosage. In contrast, the non-transgenic parental corn plants were not affected in nicosulfuron spray test. The built-in selective strategy described here is simple and convenient. To create terminable corn, any gene of interest could be inserted into the T-DNA binary plasmid reported in this study. The corn plants created can be selected and deselected as you wish by two commonly used herbicides, glyphosate and nicosulfuron. Therefore, the containment technology we report here could serves as an insurance policy for no contamination in food or feed supply.