Cloning Andanalysis Of NAC Transcription Factor Gene In Tobacco(Nicotiana Tabacum L.)

The nicotine is a secondary metabolite synthesized in the root,a tobacco-specific alkaloid. After topping, the content of nicotine accumulated, but the reason why the content increased and the regulation mechanism of nicotine are not clear. Therefore, suppression subtractive hybridization was adapted to construct a cDNA library of root tip before and after topping and screen differentially expressed genes, which has great potential to reveal the molecular basis referred to the impact of topping on the nicotine content.There are a large number of transcription factor genes in plant and they play an important role in regulating plant development and stress response. AP2/EREBP,bZIP,MYB,WRKYå'ŒNAC are common transcription factors family. In this study, NAC transcription factor EST sequences was screened out from SSH-cDNA library and it was as a probes to clone the full-length cDNA sequences of NtNAC-R1.It had an important significance on the study of molecular mechanisms in regulation of nicotine biosynthesis. The main results are as follow:(1) Real-time PCR technique was applied to verify expression patterns before and after topping of EST sequences of different gene.It showed that F-box,MYB,NAC,SAHH,ST1 had an increased expression level at 24h after topping,WRKY,AUXIN -induced mRNA had an decreased expression level at 24h after topping.(2) The full-length cDNA sequence of NtNAC-R1 was amplified by in silico cloning and RT-PCR. NtNAC-R1 had an 936bp open reading frame in length, encoding 311 amino acids, with a typical conserved domain of NAC transcription factor family.(3) Tissue-specific analysis showed that the two genes had a high expression level in roots. The expression pattern of NtNAC-R1 in root before and after tobacco topping was analyzed by RT-PCR and Northern blotting. It showed that NtNAC-R1 had a decreased expression level at 2h and 4h after topping, then rise.(4) The recombinant expression vectors of pRSETB-NtNAC-R1 was constructed and transformed into E. coli. The target proteins was successfully expressed in inclusion bodies.(5) The sense and anti-sense expression vectors of NtNAC-R1 gene were constructed and got antisense transgenic plants. The expression patterns of nicotine biosynthesis genes PMT and ODC in antisense transgenic plants were analyzed by RT-PCR. It showed that they had a decreased expression level.