Analysis Of Genes Involved In Nicotine Biosynthesis In Nicotiana Tabacum

Small RNAs are a group of regulatory molecules that play important roles in biological systems in eukaryotes by suppressing expression of target genes at the transcriptional and/or post-transcriptional level through specific base pairing with their targets. In plants, miRNAs have been demonstrated to be involved in many biological and metabolic processes, including developmental regulation, growth control, cell differentiation, signal transduction, and biotic and abiotic stresses. Over4,000miRNAs and several kinds of siRNAs have been identified in plants. However, no tobacco-specific miRNA has been experimentally isolated.As the major compounds of tobacco(Nicotiana tabacum), nicotine alkaloids have defensive functions in response to pathogen infection and herbivore wounding. Wounding is believed to induce defensive response in tobacco, but the mechanism responsible for this response is yet to be uncovered. To get insight into the role of small RNAs in damage-induced responses, we sequenced and analysed small RNA populations in roots and leaves from wounding or topping treated tobacco plants. In addition to confirmation of expression of27known miRNA families, we identified59novel tobacco-specific miRNA members of38families and a large number of loci generating phased21-or24-nt small RNAs (including ta-siRNAs). We further validated those small RNA as well as their targets through northern blot and degradome sequencing, results indicated a number of miRNAs and phased small RNAs were found to be responsive to wounding or topping treatment. The expression changes of miRNAs and phased small RNAs responsive to wounding or topping and identification of defense related targets for these small RNAs suggest that the inducible defense response in tobacco might be controlled by pathways involving small RNAs.Nornicotine is the second most abundant alkaloid in most tobacco cultivars and its conversion is mainly controlled by the nicotine N-demethylation. In our previous studies, we cloned an MTHFR (NtMTHFRl) from tobacco and applied some studies on its role whthin the pathways of tobacco nicotine biosynthesis. Based on our understandings, we sequenced the tansgenic tobacco plants through microarray and analyzed5nicotine pathway gene expression. Results indicated the overexpression or RNAi of MTHFR in tobacco didn’t altered the expression of those nicotine pathway key gene e.g. PMT and MTHFR mainly regulates the nicotine N-demethylation at the last few steps of tobacco nicotine biosynthesis.Tobacco nicotine biosynthesis is a complicated physiological metabolic process, which contains a large number of catalytic enzymes and proteins encoded by different genes. In this process. miRNA has been found to have functions on regulating its target gene and participate in such a complex physiological reaction. Furthermore, as the completion of tobacco whole genome sequence, more tobacco nicotine metabolic pathway gene will be identified, which will allow us to have a more in-depth understanding of nicotine synthetic mechanisms.