烟草中乙烯响应因子NtERF9对烟碱转化代谢途径的调控机理研究

Nicotine conversion increases the content of nornicotine and has adverse effects on human health and tobacco quality. Ethylene could induce significantly the expression level of CYP82E4, which is the primary gene of nicotine conversion metabolic pathway. However, the regulation mechanism of ethylene on CYP82E4 is still unknown. In our previous research, the cDNA library was constructed, and a protein (NtERF9) was identified to bind to the promoter of CYP82E4 by yeast-one-hybrid screening. Sequence analysis showed that this protein should be a member of ethylene response factor family (ERF) because of its AP2/ERF domain. The gene expression of NtERF9 is strongly response to ethylene. The objectives of this project are to elucidate the function and regulation mechanism of NtERF9 on CYP82E4 and nicotine conversion, and explore the potential node role of NtERF9 in the regulation of ethylene on CYP82E4. This research will enrich our understanding for the regulation network of ethylene on plant secondary metabolites, and also provide theoretic foundation and technical support for improving the quality of tobacco alkaloid using the method of metabolic engineering.

烟草中烟碱向降烟碱转化会导致降烟碱含量的累积，进而对人类健康和烟叶品质产生不利影响。乙烯能够诱导调控烟碱转化代谢途径主效基因CYP82E4的表达，但调控的分子机理尚不清楚。我们前期构建cDNA文库，以CYP82E4启动子为诱饵通过酵母单杂交技术筛选到一种与其互作的蛋白（NtERF9），序列分析表明该蛋白具有AP2/ERF功能域，推测其为乙烯响应因子家族成员。NtERF9基因还密切响应乙烯信号，本项目拟通过系统地研究NtERF9对CYP82E4和烟碱转化代谢的影响，进而揭示其调控的分子机理，并探索NtERF9在乙烯调控CYP82E4过程中可能的重要节点作用。该项目的实施将丰富乙烯对植物次生代谢调控网络的研究，为今后运用代谢工程改良烟草生物碱组分和含量提供理论依据。

生物碱是烟草中一类重要的植物次生代谢物质，但在某些烟株中会发生烟碱向降烟碱转化的现象，烟碱转化会对人类健康和烟叶品质产生不利影响。乙烯能够诱导烟碱转化主效基因CYP82E4的表达，本项目发现乙烯响应因子NtERF9与CYP82E4间存在相互作用，以此为切入点，深入研究了NtERF9对CYP82E4基因和烟碱转化代谢的调控机制。主要结果有：1）证实NtERF9与烟碱转化关键基因CYP82E4间存在相互作用；2）发现NtERF9与CYP82E4基因间存在明显的正向共表达关系；3）构建NtERF9过表达和RNAi载体并获得转基因植株，进一步分析发现NtERF9可诱导CYP82E4基因的表达和烟碱转化的发生，同时受乙烯诱导，表明乙烯可能通过诱导NtERF9基因的表达，进而调控CYP82E4基因的表达和烟碱转化代谢，转基因植株代谢谱分析还发现NtERF9基因可以影响糖类和氨基酸类的代谢；4）建立了一种白肋烟烟碱转化qRT-PCR的检测方法，为及时剔除不良转化株提供了一条新的有效途径。项目研究结果丰富了烟碱转化代谢网络，加深了对乙烯调控植物次生代谢物质的认识，为后续烟草品质改良工作提供了理论依据。