Research Specifications

Plants are frequently being located to environmental stress such as cold, drought or salinity. In response to drought and salt stresses, plants undergo molecular, physiological and metabolic changes. Usual cellular adaptation mechanism is the accumulation of osmotically active, low molecular weight, non-toxic molecules known as osmoprotectants to cope with osmotic stress. One of the most effective suitable solutes is betaine, which is not produced or accumulated in several a wide varieties of plants, animals and microorganisms. In the all betaine producers, its biosynthesis is occurred in a two-step process. In the first stage, various enzymes that catalyze the reaction depend on the organism. E.coli uses the membrane bound choline dehydrogenase (CDH) in combination with betaine aldehyde dehydrogenase (BADH) that betA encodes CDH while betB encodes BADH. In this research CDH and BADH genes from native E.coli BL21 were isolated, cloned and expressed. The total DNA was amplified by PCR using specific primers. The amplified DNA products were separated and ligated in to pTG19-T cloning vector. The E.coli DH5 was transformed using ligation reaction and transformants were selected on white-blue test medium containing antibiotic ampicillin, IPTG and X-gal. Positive vector of BADH gene and CDH gene subcloned into pBI121 plant expression vector with BamHI and SacI restriction enzymes, respectively. Agrobacteriummediated transformation was used to introduce the E. coli betA and betB genes in to plants, which do not naturally accumulate betaine.