转录因子NtMYB305通过AT-rich元件调控NtPMT表达及烟碱合成的分子机制研究

烟碱是烟草中一种最主要的生物碱，在植物防御、烟草工业及农业生产上发挥重要作用。腐胺N-甲基转移酶（PMT）是烟碱合成的关键限速酶，其基因启动子上的三个顺式作用元件（G-box、AT-rich及GCC-box-like）控制着茉莉酸（JA）对其基因表达的完全激活。近几十年来，AT-rich元件的结合因子一直未被发现。我们在栽培烟草（Nicotiana tabacum L.）中筛选到一个转录因子NtMYB305能够结合NtPMT启动子AT-rich元件并激活其表达。本项目将利用生物化学、分子生物学及遗传学方法深入解析NtMYB305在调控烟碱合成中的作用机制，阐明其响应JA并协同NtMYC2、NtERFs等G-box、GCC-box-like结合因子共同调控NtPMT表达及烟碱合成的分子机理。本研究不仅有助于进一步完善烟碱合成分子网络，同时将为实际生产中的烟草育种工作提供技术支持。

Nicotine is the major type of alkaloids in tobacco which functions as a defensive component against insect herbivores and displays an important role in tobacco industry and agricultural production. Putrescine N-methyltransferase (PMT) is the key rate limiting enzyme in nicotine biosynthesis. Studies on the promoter of PMT identified a regulatory composite known as GAG element which is composed of a G-box, an AT-rich region and a GCC-box-like element. Each of the three elements plays an indispensable role in the fully expression of PMT activated by jasmonic acid (JA). In the past decades, factors that bind to the G-box and GCC-box-like elements have been identified as bHLH family (such as MYC2) and ERF family transcription factors, respectively. However, factors binding to the AT-rich element still remain unknown. Recently, we have screened a transcription factor NtMYB305 in cultivated tobacco (Nicotiana tabacum L.) that binds to the AT-rich element of NtPMT promoter and activates its expression. In this study, we will investigate the molecular mechanism of transcription factor NtMYB305 in nicotine biosynthesis using a variety of methods involved in molecular biology, biochemistry and genetics. We will elucidate the synergetic regulation mechanism of NtMYB305、NtMYC2 and NtERFs in the process of NtPMT expression and nicotine biosynthesis. Our research will not only improve the molecular network of nicotine biosynthesis, but also provide scientific supports for the breeding of tobacco varieties in actual production.