NtNAC-R1Mediated The Process Of Tobacco Nicotine Biosynthesis Induced By Topping

The nicotine is main synthesized in the tobacco roots. After toppingof tobacco plant, tobacco roots growth enhancement, and a dramatic increase inamount of nicotine synthesis, but the reason why the content increased and theregulation mechanism of nicotine are not clear. For the study of nicotine biosynthesisof molecular regulation mechanism, in our lab, suppression subtractive hybridizationwas adapted to construct a cDNA library of root tip before and after topping andscreen differentially expressed genes. This thesis is based on the NtNAC-R1transcription factor which was screened out from SSH-cDNA library and cloned asthe research object,identification of intron and subcellular localization of the gene, bytransient expression analysis, transgenic technology analysis it’s role in hormonesignal transduction and nicotine biosynthesis and root development of the tobaccoplant. The main results are as follows：(1) RT-PCR was applied to check expressing of gene NtRNAC-R1and putrescineN-methyl transferase(PMT) in organization of root tips of plants had been treatedwithout topping and after topping24hours. The result shows that the expressionlevel of NtRNAC-R1and PMT are both increased notably.(2) NAC transcription factor NtNAC-R1was isolated by using tobacco genomic DNAand it’s two introns were identified, Size are613bp and97bp respectively, whichhas the typical splicing features “GU…AG” structure, and AU-rich, belongs to U2type intron.(3) Constructing NtNAC-R1connected with GFP fusion expression vector pS1300-NtNAC-R1.Subcellular localization experiments in tobacco mesophyll cellsindicated that NtNAC-R1was localized in the nucleus.(4) Establishing transient expression analysis system, over-expression of NtNAC-R1gene followed by RT-PCR detection indicated nicotine biosynthesis key gene PMTexpression amount increases while JA signal marker gene PDF1.2and IAAresponsing gene IAA13expression amounts decrease.Constructing NtNAC-R1gene RNAi vector IR-NAC–RI, IL-60BS virus plasmid auxiliary mediatedinjection of tobacco leaves, the RT-PCR analysis of the PMT, PDF1.2, IAA13 expression pattern indicates that the PMT gene expression level decreased,PDF1.2, IAA13expression levels increased.The above experimental results show that the tobacco NtNAC-R1transcriptionfactor involve in a variety of hormonal signals via topping, under the cross talking ofJA and IAA signal pathway, and then regulates nicotine biosynthesis.