Mechanism Of Tobacco Transcription Factor NtMYB305a In Regulating Nicotine Biosynthesis

Nicotine is an important and most abundant natural alkaloid in tobacco,which accounting for about 90% ?95% of the total alkaloid in tobacco.Putrescine is derived from plant urea cycle which is the key precursor of nicotine synthesis,and methylputrescine synthesis is catalyzed by putrescine methyltransferase PMT which is the most critical step of nicotine synthesis.Nicotine synthesis and expression of Nt PMT gene in tobacco are closely related to jasmonin(JA)signaling pathway in response to insect damage and mechanical damage.When tobacco plants were subjected to biological and abiotic stresses,such as insect pests and mechanical damage,the content of jasmonin increased sharply in the plant,and promoted the expression of nicotine synthesis gene in the root of tobacco,and increased the nicotine content of tobacco plants.Studies have revealed that JA-induced expression of Nt PMT gene is controlled by a regulatory region called GAG in the promoter,which contains three cis-acting elements,G-box,AT-rich and GCC-like.Previous studies have proved that b HLH transcription factor MYC2 and ERF transcription factor ERF189/199 can recognize and bind G-box and GCC-box-like elements within GAG regulation segment and regulate nicotine synthesis in tobacco,respectively,but the interaction factors and regulatory mechanisms of AT-rich elements remain unclear.In this study,a R2R3-MYB transcription factor Nt NYB305 a binding to the GAG regulation region in the promoter of putrescine methyltransferase gene Nt PMT1 a was screened from tobacco c DNA yeast expression library by yeast one-hybrid system.EMSA assay,chromatin immunoprecipitation(Ch IP)assay and in vitro transcriptional activation assay further demonstrated that Nt MYB305 a could bind to the ?30 bp AT-rich element within GAG region of Nt PMT1 a promoter independent of a certain number of G/C bases.This discovery reveals a novel binding mode between R2R3-MYB transcription factor and DNA cis-acting elements Gene function studies showed that Nt MYB305 a could affect accumulation of nicotine in tobacco by regulating expression levels of nicotine synthesis genes,and can coordinate with transcription factor Nt MYC2 a to regulate nicotine synthesis in tobacco.This study has important scientific value for understanding the molecular regulation mechanism of nicotine metabolism in tobacco.The main results are as follows:1.Nt MYB305 a was screened from c DNA yeast expression library and identified to interact with GAG region of Nt PMT1 a through Y1 H system.Genetic evolution analysis showed that Nt MYB305 a had a high homology with Lx S?Nt MYB305 of ornamental tobacco.The expression pattern of Nt MYB305 a show no significant difference in the root,stem and leaf of tobacco,and the expression level of Nt MYB305 a in root was significantly regulated by JA and topping.The results of subcellular localization analysis showed that Nt MYB305 a is a transcription factor located in the nucleus.2.The molecular interaction between Nt MYB305 a and GAG regulatory segment has been analyzed.EMSA analysis proved that Nt MYB305 a could bind to the AT-rich motif of GAG element in Nt PMT1 a promoter independent of a certain number of G/C bases in vitro.Furthermore,we constructed stable Nt MYB305 a overexpressed transgenic plants,and proved that Nt MYB305 a could bind to the AT-rich element of GAG region in Nt PMT1 a promoter by chromatin immunoprecipitation analysis(Ch IP-q PCR)and transcriptional activation experiments in vivo.3.The biological function of Nt MYB305 a in regulating nicotine synthesis has been proven.Nicotine content of transgenic tobacco plants were quantitatively detected in this study,compared with the control,the nicotine content of Nt MYB305 a overexpressed plants increased by about 54% before topping,while nicotine level of Nt MYB305 a RNAi tobacco plants decreased by about 53%.After toppling,nicotine content in Nt MYB305 a overexpressing transgenic tobacco plants was approximately about 35% higher than that in control tobacco plants,while nicotine content in Nt MYB305 a RNAi transgenic tobacco leaves decreased by about 46%.4.The expression regulation of Nt MYB305 a on the functional genes of nicotine synthesis was analyzed.The expression levels of several nicotine synthase genes,such as Nt PMT,Nt ODC,Nt ADC,Nt QPT,Nt A622 and Nt BBL,are regulated by Nt MYB305 a.In addition,EMSA and Ch IP experiments proved that Nt MYB305 a also recognize and bind to the specific AT-rich element of Nt ODC,Nt ADC,Nt QPT,Nt A622 and Nt BBL promoters.5.The synergistic regulation of Nt MYB305 a and transcription factor Nt MYC2 a on tobacco nicotine synthesis was studied.It was proved that Nt MYB305 a and Nt MYC2 a synergistically regulated nicotine synthesis in tobacco,however,neither yeast two-hybrid assay nor fluorescence bimolecular complementation assay demonstrated the interaction between Nt MYB305 a and Nt MYC2 a.What is more,there was no interaction between Nt MYB305 a and tobacco JAZs protein.We hypothesized that unknown regulatory factors may be involved in the molecular interaction between Nt MYB305 a,Nt MYC2 a and JAZs proteins in tobacco.In this study,the R2R3-MYB transcription factor Nt MYB305 a binding GAG region was screened by Y1 H system.In vivo and in vitro experiments proved that Nt MYB305 a could specifically recognize and bind AT-rich element in GAG segment.It was further demonstrated that Nt MYB305 a binds to ?30A/T DNA fragments in a G/C independent way,revealing a new molecular interaction between MYB transcription factor and DNA cis-acting elements.Meanwhile,the molecular mechanism of Nt MYB305 a regulating nicotine metabolism of tobacco was analyzed,which laid a foundation for the construction of molecular regulatory network of nicotine synthesis.