Roles Of MYC2 And MYB305 In Regulating Nicotine Synthesis Genes

Jasmonic acid(JA)plays a key role in the regulation of nicotine metabolism in tobacco.In order to reveal the molecular mechanism of JA-pathway mediated nicotine metabolism,we analyzed the regulatory effects of downstream transcription factors MYC2 and MYB305 on the expression of nicotine synthesis genes.The studies were carried out using the gene overexpressing plants of MYC2 and MYB305(MYC2-OE,MYB305-OE)and the corresponding gene silencing plants(MYC2-RI,MYB305-RI),the gene silencing plants of JA receptor protein gene COI1(COI1-RI)and its hybrids with MYC2-OE and MYB305-OE(COI1-RI×MYC2-OE,COI1-RI×MYB305-OE),respectively,and the control plant(TN90 tobacco).The jasmonic acid sensitivity and nicotine content of the above plants were analyzed to determine the effects of MYC2 and MYB305 on tobacco JA sensitivity and nicotine synthesis.The expression of MYC2 and MYB305 were determined in the plants overexpressing MYC2 or MYB305 and those with MYC2 or MYB305 silenced,in order to investigate the transcriptional regulation between MYC2 and MYB305.And,the expression levels of ERF-like nicotine synthesis regulators(ERF189,ERF168)were also analyzed with above plants to determine the regulatory roles of MYC2 and MYB305 on ERF genes.Subsequently,the expression of functional genes of nicotine metabolism,including Arginine decarboxylase(ADC),Ornithine decarboxylase(ODC),Putrescine N-methyltransferase(PMT),Quinolinate phosphoribosyl transferase,(QPT),were tested in the gene overexpression or silencing plants of MYC2 and MYB305 to uncover the roles of MYC2 and MYB305 in regulating these gene.Furthermore,the JA sensitivity,nicotine content and expression of nicotine synthesis genes were analyzed in the hybrid progenies of COI1-RI with MYC2-OE and MYB305-OE to study the roles of MYC2 and MYB305 in restoring the nicotine synthesis capability of COI1-silenced plants.The promoters of ADC and ODC genes,which were negatively regulated by MYC2 upon JA treatment,were cloned and their GUS reporter vectors were constructed for further studies.The main findings of this study are as followings:1.The effects of MYC2 and MYB305 on tobacco JA sensitivity and nicotine synthesis: The results showed that the transcription factor MYC2 could enhance the sensitivity of tobacco to JA signals and positively regulate the synthesis of nicotine;MYB305 participated in the regulation of nicotine synthesis,but did not affect the JA sensitivity of tobacco.2.The transcriptional regulation between MYC2 and MYB305 and their regulation on nicotine metabolism-related ERF genes: The results showed that the transcription factor MYC2 up-regulated the expression of MYB305,but the transcription factor MYB305 did not up-regulate the expression of MYC2.MYC2 positively regulated the expression of ERF189 and ERF168,and MYB305 participated in the expression regulation of ERF189 and ERF168.3.Roles of MYC2 and MYB305 in regulating the nicotine metabolism functional genes: The results showed that overexpression or gene silencing of MYC2 and MYB305 both up-regulated the expression of nicotine synthesis genes PMT and QPT;after overexpressing MYC2,it could strongly inhibit the expression of ADC and ODC induced by JA;overexpression or silencing of MYB305 down-regulated nicotine expression of synthetic genes ADC and ODC.4.The roles of MYC2 and MYB305 in restoring the nicotine synthesis capability of COI1-silenced plants: The results showed that overexpression of MYC2 or MYB305 could not restore the JA sensitivity and nicotine synthesis of COI1-RI tobacco.But,overexpression of MYC2 could restore the expression of nicotine metabolism function genes PMT and QPT in COI1-RI tobacco to a certain extent,but it could not restore the expression of ADC and ODC;overexpression of MYB305 could restore the nicotine metabolism function genes PMT and QPT in COI1-RI tobacco,partially restored the expression of ADC and ODC.5.Construction of the GUS reporter vectors of ADC and ODC promoters: In order to reveal the molecular mechanism of MYC2 overexpression inhibiting the expression of JA-induced ADC and ODC,the promoters of ADC and ODC genes were cloned,and the vector for expressing the fusion of the promoters of ADC,ODC and the GUS gene was constructed by Gateway LR recombination technology,the transgenic materials were cultivated by leaf disc transformation method.This study proved that MYC2 and MYB305 were involved in the expression regulation of nicotine metabolism function genes and regulatory genes,which provided theoretical basis for revealing the molecular mechanism of the regulation of the nicotine synthesis gene by the jasmonic acid pathway;we constructed the protein expression vector of the ADC and ODC promoter gene fusion GUS,and cultivated its transgenic plants,which provided the material basis for the subsequent molecular regulation mechanism research.