转录因子NtMYC2b参与烟草烟碱含量杂种优势形成的机制研究

Transcription Factor NtMYC2b Is Involved in the Formation of Heterosis of Nicotine Content Trait

为探讨bHLH转录因子家族成员MYC2b基因与烟叶中烟碱含量及烟碱含量杂种优势表现的相关性,利用RT-qPCR分析NtMYC 2 b基因与烟碱合成途径关键酶基因的表达,测定烟碱含量和烟碱杂种优势表现。结果表明,移栽后80 d,NtMYC 2 b在烟草亲本GDH88和巴斯玛根部的表达量较移栽后66 d上调,其根和叶中烟碱含量增加,杂交种根中NtMYC 2 b、鸟氨酸脱羧酶基因(ODC)、精氨酸脱羧酶基因(ADC)、N-甲基转移酶基因(PMT)、N-甲基腐胺氧化酶基因(MPO)的表达量相对于亲本平均表达量均明显上调,且除MPO外,NtMYC 2 b与ODC、ADC、PMT、QPT(喹啉酸磷酸核糖转移酶基因)在强优势组合VA116×巴斯玛中的相对表达量均明显高于弱优势组合VA116×GDH88,根和叶中烟碱含量及其杂种优势表现也具有相同的趋势,表明NtMYC 2 b参与了烟碱的合成,还可能参与了烟碱含量杂种优势的形成。相关性分析表明,在亲本根部,NtMYC 2b表达量与PMT表达量呈显著正相关;在杂交种根部,NtMYC2b表达量与ADC、MPO表达量呈显著正相关;在杂交种叶片中,NtMYC2 b表达量与烟碱含量杂种优势呈显著正相关,PMT表达量与ODC、ADC表达量均呈显著正相关。关键酶基因启动子序列分析表明,ADC中含有1个MYC结合位点和1个G-box元件;PMT中含有7个MYC结合位点和1个G-box元件。推测NtMYC 2 b可能通过与ADC、PMT的顺式元件MYC结合位点或G-box元件特异性结合调控ADC、PMT的表达,进而促进烟碱的合成,且NtMYC 2 b还参与了烟碱含量杂种优势的形成。

To investigate the correlation between transcription factor MYC2b and nicotine content and its heterosis in tobacco,RT-qPCR was used to analyze the expression of NtMYC 2 b and key enzyme genes in the nicotine synthesis pathway,and the nicotine content and its heterosis in roots and leaves of tobacco were determinated.The results showed that the expression of NtMYC 2 b in parent roots(GDH88 and Basma)was upregulated at 80 d compared with 66 d after transplanting,and their nicotine content in root and leaves was also increased.The expressions of NtMYC 2 b,ornithine decarboxylase gene(ODC),arginine decarboxylase gene(ADC),putrescine-N-methyltransferase gene(PMT)and N-methylputrescine oxidase gene(MPO)were significantly upregulated in root of hybrid relative to their parents.The relative expression levels of NtMYC 2 b,ODC,ADC,PMT and QPT(quinoline phosphoribosyltransferase gene)in the strong heterosis combination(VA116×Basma)were significantly higher than those in the weak heterosis combination(VA116×GDH88),meanwhile,the nicotine content and its heterosis also showed the same trend in roots and leaves of hybrid.It indicated that NtMYC 2 b was involved in the synthesis of nicotine and might also be involved in the formation of heterosis of nicotine content.Correlation analysis showed that the expression of NtMYC 2 b was significantly positively correlated with PMT in the parent roots,the same with ADC and MPO in hybrid roots.Moreover,between NtMYC 2 b expression and nicotinic content heterosis,between PMT expression and ODC and ADC expression,was also a significant positive correlation in hybrid leaves.Sequence analysis showed that the promoter sequence of ADC contained 1 MYC binding site and 1 G-box element,the promoter sequence of PMT contained 7 MYC binding sites and 1 G-box element.Therefore,it is speculated that NtMYC 2 b may regulate the expression of ADC and PMT by specifically binding with cis-element MYC binding site or G-box elementto promote the synthesis of nicotine.These findings suggest that NtMYC 2 b is involved in the synthesis of nicotine and the formation of heterosis in nicotine content.