Cloning And Function Analysis Of Choline Monooxygenase(AcCMO) From Atriplex Canescens

Several environmental factors adversely affect plant growth and development andfinal yield performance of a crop. Drought, salinity, nutrient imbalances (includingmineral toxicities and deficiencies) and extremes of temperature are among the majorenvironmental constraints to crop productivity worldwide. It is estimated that lessthan10%of the world’s arable lands may be free of major environmental stresses,with drought and salinity stresses being the most widespread. For instance, worldwidearea mapped as being affected by salinity is more than3×106km2, or approximately6%of the total land area.Glycine betaine(GB) is an amphoteric compound that is electrically neutral over awide range of physiological pH values. Studies in vitro have indicated that GB is notmerely a nontoxic, cellular osmolyte that raises intracellular osmolarity when a cell isexposed to stress-induced hyperosmotic conditions: it has been well documented that,in vitro, GB stabilizes the structures and activities of enzymes and protein complexesand maintains the integrity of membranes against the damaging effects of excessivesalt, cold, heat and freezing. Moreover, the gene CMO is the key enzyme in thesynthesis of GB.In this article, Atriplex Canescens(called saltbush) was selected selected as thematerial which we were studying. After comparing with CMO sequences of relatedspecies in the NCBI, degenerate primers were designed for amplification. Extractionof RNA from Atriplex Canescens, treated with cold, was performed. Subsequently,PCR was carried out after reverse transcription, and the AcCMO gene sequence ofAtriplex canescens was obtained, which contained1317bp, encoding438amino acids.We submitted the sequence to Genbank, and obtained a accession number-JF776158.Evolutionary analysis indicated that AcCMO of Atriplex canescens was closest tothose of Atriplex nummularia and Atriplex prostrata.In order to predict the function of AcCMO gene, we transferred it to E. coli and yeast for expression. In yeast cells, we treated the yeast with salt, alkali, drought,oxidation, high temperature and low temperature stress. The results showed that theAcCMO gene could improve the tolerance of yeast in the salt, alkali, drought and lowtemperature stress. It indicated that AcCMO was related to resistance to salt, alkali,drought and low temperature stress in plant, which might due to that betaine couldplay a role in a variety of stress.To further confirming the function of AcCMO gene in plants, the recombinantplasmid of AcCMO gene were transformed into Agrobacterium and then transformedinto Arabidopsis thaliana. A total of two transgenic seedlings were obtained. Thebasis for function analysis of AcCMO gene in Arabidopsis thaliana next wasconstructed.