Cloning And Analysis Of WRKY-like Gene In Tobacco(Nicotiana Tabacum L.)

Nicotine is the unique secondary metabolite in the synthesis of tobacco root..In the period of tobacco production, content of nicotine in tobacco increased dramatically after topping. There are reports that the reason why the ability of nicotine synthesis strengthens may be due to damage or the change of plant endogenous hormones or the development of the root.For studying the reason why the content increased and the regulation mechanism of nicotine,in our lab, WRKY 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 NtWRKY-R1. It had an important significance on the study of molecular mechanisms in regulation of nicotine biosynthesis. The main results are as follow:1. The clone and bioinformatic analysis of WRKY-likea.The full length cDNA of NtWRKY-R1was obtained. NtWRKY-R1contains an open reading frame of915bp and encoding304amino acids.b.Bioinformatics analysis indicated that the NtWRKY-R1of tobacco has the closest relationship with tomato(Solamtm lycopersicum).The protein encoded by NtWRKY-R1was a soluble protein and has a typical WRKY conserved domain and a C2H2zinc finger domain. It was belonged to group Ⅱ of WRKY transcription factors and was predicted a nuclear localization signal. Study showed that the tobacco NtWRKY-R1contains two introns, which has the typical splicing features "GU... AG" structure, and the intron1belongs to R type intron.2. The analysis of the expression of NtWRKY-R1a.Tissue-specific analysis showed that NtWRKY-R1had a high expression level in root.b.The analysis of the expression of NtWRKY-R1before and after topping by Northern, the expression of NtWRKY-R1after topping lower than before.c. The expression pattern of NtWRKY-R1in root before and after tobacco topping was analyzed by RT-PCR and Northern blotting. It showed that NtWRKY-R1had a decreased expression level at2h-4h after topping and rise at6h,then decrease in10h-24h.3. The build of PROK2-NtWRKY-R1expression vector and analysis of transient expression The sense expression vectors of PROK2-NtWRKY-R1gene were constructed and transform into tobacco protoplasts for transient expression. The result of RT-PCR: PDF1.2、PMT1had a decreased expression level and NPR1、NtWRKY-R1has a increase expression level.4. The clone and bioinformatic analysis of PMT1promotorClone the PMT1promotor from the root of the tobacco by RT-PCR.The result of the bioinformatic analysis of PMT1promotor shows that PMT1promotor include three W-box Cis-acting element and several Cis-acting element relative to hormone.All of these results show that NtWRKY-R1involve in JA and SA pathway;NtWRKY-R1suppress the express of PMT1,PMT1promotor include three W-box Cis-acting element.Presumably,NtWRKY-R1may involve in the molecular mechanisms in regulation of nicotine biosynthesis.These results provide some evidences for the research that NtWRKY-R1responses to the damage to overground part and relates with the enhance of ability of nicotine synthesising after topping.