Cloning of Sorghum bicolor Chloroplast Transit Peptide (CTP) of 5-enolpyruvylshikimate-3-phosphate Synthase(EPSPS) and Its Functional Validation in Transgenic Maize(Zea mays)
Abstract：5-enolpyruvylshikimate-3-phosphate synthase(EPSPS), which has an active site with glyphosate, can catalyze 5-enolpyruvylshikimate-3-phosphate(EPSP) synthesis with shikimate-3-phosphate(S3P) and phosphoenolpyruvate(PEP), and the enzyme activity can be inhibited when combine with glyphosate which are important in herbicide resistance genetic engineering of plants. To cultivate the maize(Zea mays) having resistance to glyphosate, we cloned Sorghum bicolor chloroplast transit peptide (CTP) of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. Expressional vectors were constructed after fused the CTP and Agrobacterium sp. strain CP4 EPSPS gene together, while using Ubiquitin as promoter and 35S polyA as terminator. The vectors were transformed into maize, with vectors that did not contain CTP fragment as control. Analysis of the transgenic plants by PCR, Southern blot and ELISA showed that: Although the gene expression level of plants from CP4 EPSPS and CP4 EPSPS with CTP was almost the same, plants having the CP4 EPSPS without CTP did not have resistance to glyphosate. On the contrary, plants having the CP4 EPSPS with CTP had strong resistance to glyphosate. These results indicated that CTP did not affect the expression of CP4 EPSPS, but played an important role in the resistance of glyphosate. In addition, we confirmed that the CTP we cloned had the right biology function. In this study, we provide a framework and inspiration for cultivating the crops with resistance to glyphosate by EPSPS.
