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Nicotine is a secondary substance synthesized in tobacco roots.In flue-cured tobacco planting,tobacco decapitation is an effective practice to promote nicotine biosynthesis by regulation of the redistribution of total nitrogen amounts,However,proteins relevant to nicotine synthesis in tobacco roots has not been identified and characterized yet.It is important to explore the regulation of nicotine biosynthesis in tobacco roots.To identify the proteins relevant to nicotine synthesis,the protein patterns in roots of flue-cured tobacco (cv.K326) before and after decapitation were analyzed.In the present study,the protein patterns in roots of flue-cured tobacco were analyzed by two-dimensional electrophoresis (2-DE),and the differentially-expressed spots were identified by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Paired comparison of 2-DE maps revealed 26 spots of differentially-expressed proteins in roots before and after decapitation.Furthermore,nine differentially-expressed spots were identified.There were four proteins which were enzymes possibly involved in nicotine biosynthesis.In addition,the roles of the four enzymes in nicotine biosynthesis were discussed in a putative network.Our results would contribute to the understanding of the regulation pathway of nicotine biosynthesis and further to the molecular manipulation on the nicotine contents in flue-cured tobacco.