Expression Of SeCMO And SePEAMT Genes In E.coli And Drought Tolerance Of Transgenic Tobacco With SePEAMT+SeCMO Gene

Betaine will be accumulated in plants in response to drought and salinity stress.The betaine accumulation plays an important role in improving osmotic adjustment and protecting cell membrane,therefore,betaine is a very effective osmoprotectant.Choline monooxygenase(CMO) and Phosphoethanolamine N-methyltransferase(PEAMT) are the key enzyme in the pathways of GlyBet synthesis in plants.The genes related to glycine betaine synthesis are transformed into plants through genetic engineering,which could enhance the drought tolerance of plants.The SeCMO and SePEAMT genes were subcloned into the expression vector pGEX-6p1, recombinant plasmids pGEX6p1-SeCMO and pGEX6p1-SePEAMT were obtained and transformed into Escherichia coli BL21(DE3).Expression induced by IPTG and SDS-PAGE analysis showed that the SeCMO and SePEAMT gene could express in E.Coli.The fusion proteins were purified with affinity chromatography of GST.To evaluate the drought tolerance of the transgenic tobacco,the wild type(WT) and transgenic tobacco were exposed to drought stress by withholding water.During the stress period,the Glycinebetaine(GB) content and other physiological parameters including relative water content(RWC),photosynthesis,cell membrane stability,active oxygen metabolism and osmoprotectants content of tobacco plants were measured.The transgenic tobacco plants accumulated significantly higher levels of GB than WT tobacco plants.Under the water stress, the transgenic lines had higher RWC than WT tobacco plants,and the net photosynthesis rate (Pn) and stomatal conductance(Gs) of all plants were reduced,while PC26,PC45 had significantly higher Pn than WT plants and lower substomatal CO₂ concentration(Ci) than non-stress plants,which demonstrating that the photosynthetic system damaged slightly.The percentage of ion leakage and malondialdehyde(MDA) content of transgenic plants were lower than those of the WT plants,while the SOD,POD activity and content of proline, soluble sugar,soluble protein of transgenic plants were generally higher than those of WT plants.Results convinced that the higher level GB content of transgenic tobacco may play an important role in osmotic adjustment and protecting membranes and enzymes from damage, thus the drought tolerance of transgenic tobacco is enhanced.