Expression And Characterization Of Mannose-6-phosphate Isomerase From Escherichia Coli DH10B And Resistance Analysis Of Transgenic Potato Harboring Cry3A

Mannose-6-phosphate isomerase (PMI) is encoded by the manA gene from Escherichia coli DH10B as a selectable marker which was evaluated for the transformation of plant. It converts mannose-6-phosphate to fructose-6-phosphate allowing for selection of transgenic plants with mannose as exclusive carbon source. Plants express pmi gene can use mannose to proceed regular development and metabolism while plants without pmi gene are dead owing to lacking of carbon source.Therefore, seeking pmi gene from different source and use it efficiently is a significant task for transgenic plants.The full length DNA of pmi gene (1200bp; Genbank accession no.CP000948) was cloned from Escherichia coli DH10B by PCR and used for construction of expression vectors for both prokaryotes and eukaryotes, which are pET-23b-pmi and pCAMBIA2301-pmi, respectively. Upon induction with IPTG the former one pET-23b-PMI fusion protein was express in Escherichia coli BL21 and purified through affinity chromatography with Ni+ column. We optimized the concentration of imidazole in lysis buffer and wash buffer to identify the single band.Then Western blot reconfirmed the PMI protein was about 46kD.According to the phenomenon of reaction between fructose, resorcinol and hydrochloric acid, the induced and purified PMI protein are added into mannose-6-phosphate to catalyzed the reaction at temperature of 37℃. The red complex resulting from the reaction after 7 minutes, at 100℃. This result indicated that PMI has catalyzed the conversion of mannose-6-phosphate to fructose-6-phosphate with high efficiency.We construct pmi gene into pD513, which conclude the promoter CaMV35S. Then the reading frame pD513 with pmi and CaMV35S insert into plant expression vector pCAMBIA2301. The plant expression vector transformed tobacco, and then the PCR analysis of tobacco transformants showed the integration of pmi gene into tobacco genome. These results indicated that pmi gene we cloned has the highly activity and it can be directly used in plant transformation.