Studies On Metabolic Regulation Of Tropane Alkaloids In Tobacco Through RNA-mediated Gene Silencing

Tropane alkaloids are important secondary metabolites in tobacco (Nicotiana tabacum).Studies on regulation modes of tropane alkaloids metabolism through genetic engineering,are academically important to enrich knowledge pool of metabolic regulation,and have potential value of application on reducing harm of cigarette on human health.These studies can also improve the pharmaceutical value of the tobacco tropane alkaloids in pharmaceutics,and enhance tobacco' s resistance to insects. However,this field has yet to be studied in China,and only a few publications related to this field around the world provided limited messages due to restrictions such as amounts of samples and intentions of studying.Here tobacco lines with a series of reduced nicotine levels were obtained via RNA-mediated gene silencing and through extending amount of sample,and a new kind of regulation mode of tropane alkaloids metabolism was found related to nicotine biosynthesis and anatabine,a kind of product of its branched pathway.Genes encoding putrescine N-methyltransferase(PMT),the key enzyme controlling nicotine biosynthesis,were chosen to be targets to alter nicotine concentrations in tobacco.The gene family encoding this enzyme is constituted by five genes.Conserved sequence of 540bp which is near 3' termini of PMT was selected to be target of silencing.Three kinds of RNA-mediated gene silencing vectors were constructed,that is,vector of cosuppression,that of antisense and that of RNA interference,which were transformed to tobacco leaf plates via Agrobacterium tumefaciens mediated transformation system.After screening and confirming,645 independent transgenic tobacco lines were generated,among which data of nicotine levels were obtained from 217 lines.The phenotype of the transgenic tobacco lines appears to be normal as compared to the control plants.RNAi approach was proved to be the most efficient way to reduce the levels of nicotine,whereas cosuppression and antisense approaches were less effective.The tobacco line of the most distinct silencing were obtained via RNA interference,whose nicotine level is 3.27%compared to CK line. Nicotine level of the tobacco line of the most distince silencing obtained via cosuppression and antisense is 14.37%and 7.91%compared to CK line respectively.As to cosuppression lines,nicotine contents were reduced between 9.10%and 85.63%;as to antisense lines,between 12.39%and 92.09% RNAi lines,between 29.00%and 96.73%.Nicotine levels of these lines distributed averagely.Results of RT-QPCR showed that there is positive correlation between nicotine contents and expression of PMT,and reduction of nicotine is not as evident as depression of PMT expression.Nicotinic acid is the precursor of both anatabine and the pyridine ring of nicotine,whose synthesis is regulated by quinolinate phosphoribosyltransferase(QPT).Down regulation of PMT activity in Nicotiana would be expected to lead to elevated levels of anatabine along with reduction of nicotine levels.In this study,tobacco lines with silenced PMT showed an intriguing mode for variety of anatabine levels. Reduction of the level of nicotine does not result in elevation of anatabine levels until nicotine concentration were lower than 3mg/g, which is about 10%compared of that of CK,when a dramatically elevated anatabine level were observed.Anatabine level of the most notable line was 2.1771mg/g.Results of RT-QPCR showed no distinct change of QPT along with elevation of anatabine levels.