The Study On The Function Of NtWRKY-R1 5'UTR And NtNAC-R1 In Tobacco Root

Root is the major site of nicotine synthesis.Previous study found that tobacco topping would enhance the ability to synthesize nicotine,but the mechanism remained unclear.In order to study the signal transduction mechanism of enhance the ability to synthesize nicotine in root by topping,the two transcription factor gene of NtNAC-R1 and NtWRKY-R1 were screened from SSH-c DNA library that was constructed before and after topping in tobacco root.In this study,we investigated the role of NtNAC-R1 and NtWRKKY-R1 for inducing nicotine biosynthesis process in root after tobacco topping.The results are as follows.1.The interference expression vector(RNAi)of NtNAC-R1 was constructed and transformed into tobacco K326,and the F1 generation of tobacco plant was obtained.Using tobacco K326 varieties as materials which had been transplanted for 30 days with wild type as control group,then q PCR was performed to assess the expression level of PMT,NtWRKY-R1 and NtNAC-R1 for NtNAC-R1 over expression tobacco group and F1 generation of RNAi NtNAC-R1 transformed tobacco group.Comparing with wild type the results showed PMT expression levels of NtNAC-R1 over expressing tobacco plants were1.65 times of the control,while the PMT expression level of NtNAC-R1 RNAi tobacco plants were 0.27 times of that of the control.It may suggest that NtNAC-R1 was involved in the process of nicotine synthesis induced by topping.0.25 times and 0.17 times expression levels of NtWRKY-R1 in NtNAC-R1 over expression plant and NtNAC-R1 RNAi plant respectively compared to wild type may indicate NtNAC-R1 had no significant effect on the expression of NtWRKY-R1.2.We constructed the vector with deletion of 5'UTR,and we find it can regulate expression of gene through transient expression assay.All the results we got lay a foundation for further research on the effect from genes in regulating the biosynthesis of nicotine in roots in consequence of topping.