Studv On The Regulation Of Nicotine By G-box Element In PMT1 Promoter

Nicotine is a unique natural alkaloid in tobacco.With the deep research of nicotine,the development of nicotine has gradually applied to many fields such as chemical industry,medicine,food and health care.The demand of market for nicotine is increasing,especially for high-purity nicotine.However,the nicotine content in dry weight of the tobacco leaves is approximately 0.6-3.0%.High-nicotine varieties are essential for the development and utilization of nicotine.The biosynthesis of nicotine is inseparable from the jasmonic acid pathway.The transcription factor involved in nicotine biosynthesis,which is released by the action of jasmonic acid signaling factor,binding the G-box element in the promoter of PMT(Putrescine N-methyltransferase)for regulation of the nicotine biosynthesis.Also,PMT is a rate-limiting enzyme in nicotine biosynthesis.In this study,we cloned the PMT1 promoter and the G-box element in the PMT promoter to design three types PMT1 promoter expression vectors with one G-box,four G-box and ten G-box.Three promoters were inserted into pCAMBIA-NPT-GUS instead of 35S promoter to drive GUS gene,the resulted plastmids were designated as pC AMBIA-NPT-PMT1-1N-Pro-GUS(1N-GUS),pCAMBIA-NPT-PMT1-4N-Pro-GUS(4N-GUS),and pCAMBIA-NPT-PMT1-10N-Pro-GUS(10N-GUS).Furthermore,PMT1 was inserted behind these promoters for expression.Our studies will provid a new research method for nicotine production.Themain results were listed as following aspects:First,we cloned a 607 bp PMT1 promoter sequence from Nicotiana tabacum L.K326,and analyzed its response components.Sequence analysis revealed that the obtained PMT1 promoter sequence showed high similarity to that of N.sylvestris.Using Plant-CARE promoter database,we predicted that the sequence contains promoter core elements TATA-box and CAAT-box,G-box,GC-motif and elements response to light,jasmonic acid,gibberellin,and heat.Next,in order to study the effect of G-box element on the function of the PMT1 promoter.we designed three PMT1 promoters containing one G-box,four G-box and ten G-box to drive the GUS gene expression,the resulte dvectors were designed as:1N-GUS,4N-GUS,and 10N-GUS.Agrobacterium-mediated method was used to infect tobacco,and transgenic plants were obtained.The obtained promoter transgenic lines were identified by DNA and GUS staining.DNA identification results showed that three vectors were successfully integrated into the tobacco genome.GUS staining showed that the leaves and roots of the three promoter transgenic plants could be stained blue,and the staining of the roots was deeper.Comparing the staining of three promoter transgenic plants,we found that 10N-GUS stained the deepest,followed by 4N-GUS,and 1N-GUS stained the lightest.GUS quantification and GUS enzyme activity results were consistent with GUS staining results.10N-GUS was the strongest,followed by 4N-GUS,then 1N-GUS.GUS staining results and expression analysis of GUS indicated that increasing the number of G-box sequence could strengthen the activity of the PMT1 promoter.Finally,in order to study the effect of G-box sequence doubling on nicotine synthesis,primers were designed based on the PMT1-mRNA(GenBank accession number:AB004322)sequence.The Nicotina tabacum L.K326 cDNA was used as a template to carry out PCR amplification,and 1162 bp PMT1 gene sequence was obtained.Sequence analysis revealed that the cloned PMT1 gene sequence is very similar to the PMT1-mRNA sequence.The cloned PMT1 gene sequence was substituted for the GUS gene in the three promoter expression vectors obtained.And three PMT1 gene expression vectors were constructed:1N-PMT1、4N-PMT1 and 10N-PMT1.Agrobacterium-mediated method was used to infect tobacco,and transgenic plants were obtained.The nicotine content of the transgenic positive plants were then determined.The results showed that the nicotine content of the three transgenic plants are significantly higher than the control,at least twice as high as the control.The nicotine content of the partial 4N-PMT1 and 10N-PMT1 strains are more than two times higher than the control,and the highest was 2.5 times.Chemical composition test found that the total and reducing sugars of the transgenic lines decreased significantly compared to the control,and the potassium content did not change significantly.The expression levels of nicotinic transcription factors MYC2a,MYC2b and PMT1 were significantly increased compared with the control,and the expression levels of PMT1 gene in the three transgenic tobaccos were not significantly different.