The Effects Of Nicotine Tobacco

1483 Words null Page
Introduction
Tobacco use is one of the major threats to human health which kills nearly 6 million people each year with an estimated 8 million deaths in the year 2030. More than 2,500 chemicals are found in tobacco and its smoke, among which terpenoids and alkaloids are the major groups [1]. Though there is clinical uncertainty about the carcinogenic property of nicotine, evidences have proved nicotine as the main psychoactive agent responsible for the development of tobacco dependence [2, 3]. Nicotine diffuses readily into brain tissue where it binds to nicotinic acetylcholine receptors reinforcing addiction. It is estimated that 3-5 mg/day is the threshold level that readily establishes and sustains addiction among smokers [4]. The LD50 of
…show more content…
Achieving this by physical and chemical processing of tobacco is not cost effective [5, 6, 7]. Therefore, developing low nicotine tobacco varieties would be a viable strategy to bring down nicotine level in tobacco products. Being a non-edible crop, genetically engineered low nicotine tobacco would be more acceptable than transgenic crop plants. Nicotine content in tobacco can be effectively reduced by manipulating the expression of the genes that are involved in nicotine biosynthesis. The nicotine biosynthesis pathway and the genes involved are well documented in Nicotiana tabacum [8, 9]. Putrescine N-methyl transferase (PMT, EC 2.1.1.53) encoded by PMT genes catalyzes the S-adenosylmethionine dependent N-methylation of putrescine to N-methyl putrescine, the first committed step in the nicotine biosynthesis in tobacco [8, 10]. PMT is a multigene family consisting of five genes (PMT1 to PMT5) of which PMT2 is the major one which is expressed in the roots of tobacco [11, 12, …show more content…
First strand cDNA was synthesized with DNase I treated RNA and antisense primers for the reference gene (β-ATPase), target genes (PMT1/5, PMT2/4, PMT3 and HCT) (supplementary table 1). In a total reaction volume of 30µl, RNA (3µg) dNTP’s (10mM) antisense primers (5picomoles) and DTT (5mM) were added and denatured at 65oC for 5minutes. Then, the samples were plunged into ice. To this reaction mix, 50 Units of reverse transcriptase (MuMLV) and reverse transcription assay buffer (5X) was added and incubated at 30ºC for 5 minutes. First strand cDNA was synthesized at 42ºC for 60minutes. Thereafter, the abundance of the PMT members and HCT was studied using gene specific sense primers by qPCR (Light Cycler LC480, Roche Diagnostics). Sense and antisense primers were designed using IDT. Sense primers specific for genes and antisense primers for a conserved region of the PMT genes. Quantitative PCR was performed in a 20µl reaction using Light Cycler 480 SYBR Green I master mix as per the manufacturer’s protocol. Previously, individual primer pairs were validated for PCR efficiency using the slope obtained in qPCR. All experiments were carried out in

Related Documents