| 5-enolpyruvlshimimate-3-phosphate synthase (EPSP synthase) is a key enzyme when synthesizing the aromatic amino acids in prokaryote, yeasts, fungi, apicomplexan parasites, plants and algae. As a necessary key enzyme of the shikimate pathway, EPSPS convert phosphoenolpyruvate and shikimate 3-phophate to 5-enolpyruvlshimimate-3-phosphate and phosphate. Glyphosate is a foliar applied, broad spectrum, non-selective,post-emergence, low-toxicity organic phosphorous herbicide capable of controlling annual and perennial grasses and dicotyledonous weeds. Due to the similarity structure between glyphosate and phosphoenolpyruvate, glyphosate can inhibit EPSP synthase peculiarly by rivalrously uniting EPSP synthase with phosphoenolpyruvate. Plants can obtain high doses glyphosate tolerance if the enzyme of this gene is overproduced and accumulated or herbicide-insensitive enzymes produced due to some amino acid mutation in active sites. For the aim of extending the application of the glyphosate and reducing the cost of herbicide,scientists cloned the herbicide-insensitive epsps genes which have the special base pair mutation and transduct them into several kinds of crops, which gains outstanding effects.As more and more various genetically modified crops appear in international market by trade,The security of the them gets much more attentions. There are a lot of disputes about them in countries of all kinds. Some states such as Japan constitute new regulations in point to keep a strict hand over genetically modified foodstuff. Genetically modified roundup-ready soya bean was invented by the American Monsanto Company firstly and got authorization to be applied in America at the same time. Till last year, half of soya beans are genetically modified roundup-ready soya bean in the world. Lately most of ten million soya beans we import are genetically modified. At present, we don't have a standard way to detect genetically modified roundup-ready products when they enter Chinese market in a great deal without labeling and claiming, Which makes it urgent and important for us to constitute and consummate a detection way. Nowadays, domestic way to detect the genetically modified roundup-ready soya bean is in the level of gene by PCR which spend one hour at least and have a high false probability. We have to use the primes to the special gene because no universal primes is available and the PCR way do need special chemical apparatuses and a good condition, which it is hard to popularize in the grass roots. The way overseas to detect the genetically modified roundup-ready soya bean is in the level of protein by ELISA or the colloidal gold immunostrip which depends on the monoclone antibody contraposing to the mutation in the No. 101 amino acid. The test is easy to finish in five minutes and inspectorate needn't have special chemical apparatuses, which is applied widely in the world. We amplified the target mutation gene from genetically modified roundup-ready soya bean genome by PCR, and then cloned it into the prokaryotic expression vector pQE30 and pET32(a). The recombinant vectors were used to transform and expressed in E.coli SG13009 and BL21 (DE3) under induction of IPTG. After purified by Ni2+-NTA and renatured through dilution renaturation, the purity of the recombinant protein is above 90%. The antigenicity of recombinant proteins was recognized by the CP4-EPSPS colloidal gold immunostrip offered by Agdia Company. So the recombinant protein can be used to production of the monoclone antibody contraposing to the mutation in the No. 101 amino acid and lays the solid foundation for a quick domestic detection kit of CP4-EPSP synthase from genetically modified roundup-ready soya bean.Aim To construct two recombinant vectors containing gene encoding CP4-EPSP synthase from genetically modified roundup-ready soya bean and express in E.coli SG13009 and BL21(DE3).Methods The target gene encoding CP4-EPSP synthase was amplified from genetically modified roundup-ready soya bean genome by PCR. Aft... |
