生物碱是烟草中一种主要次生代谢产物，主要包含烟碱、降烟碱、假木贼碱和新烟草碱四类。其中烟碱，又称为尼古丁，是烟草中最主要生物碱，占总量的95%左右。对尼古丁合成途径中的关键酶基因，如腐胺-N-甲基转移酶基因（PMT）及喹啉酸磷酸核糖基转移酶基因（QPT）进行转录水平调控是改变尼古丁含量的有效途径，而这些基因都受到茉莉素（JA）的诱导而表达上调。茉莉素诱导尼古丁合成的分子机制简述如下：当植物受到外界胁迫时，JA及其类似物含量升高，Jasmonate ZIM-domain（JAZ）蛋白被JA受体COI1招募，随后被泛素化及蛋白酶体降解。释放出MYC2转录因子结合到PMT或QPT启动子的G-box激活其转录。另一方面，MYC也能激活ERF转录因子的转录，后者结合到PMT或QPT启动子GCC-box上，随后协同激活PMT及QPT等基因的转录。另外，MYC也能结合到JAZ启动子激活其转录，从而达到反馈抑制的作用。大量研究表明茉莉素信号通路中的关键组分COI1与NtJAZ等是尼古丁合成的重要调控因子，因此解析茉莉素信号通路中的新组分可以进一步阐明尼古丁合成调控的新机制。本实验室前期筛选得到了与烟草NtJAZ1互作的蛋白NtAIDP1（AT-rich interaction domain-containing protein 1），进一步证明AIDP1可以结合NtPMT1a启动子的GAG片段的AT-rich基序以激活其转录活性。然而，AIDP1对尼古丁合成的具体调控机制并不清楚。

本论文首先通过荧光素酶互补实验验证了NtAIDP1与茉莉素信号通路中关键组分NtJAZ1、NtMYC2a及NtERF115的互作情况。结果表明，烟草瞬时表达体系中NtAIDP1与NtJAZ1、NtMYC2a与NtERF115均有互作，且互作发生在NtAIDP1的C端，而与其N端不发生互作。其次，构建了过表达NtAIDP1的转基因四倍体烟草，测定了过表达植株中尼古丁合成相关基因的表达情况。结果表明，在三株独立的NtAIDP1过表达四倍体烟草植株中NtPDF1.2和NtJAZ1的表达水平显著高于野生型中的表达水平，而NtPMT1a、NtQPT2和NtMYC2a的表达水平与野生型的表达水平没有差异。这些结果暗示过表达NtAIDP1不影响四倍体烟草中尼古丁含量。随后，利用CRISPR/Cas9技术构建二倍体本氏烟草nbaidp1突变体，并筛选得到了一株纯合突变体#44，检测了该突变体中尼古丁合成相关基因的表达水平和尼古丁含量。结果表明，与野生型植株相比，nbaidp1植株表现出MeJA诱导的NbJAZ1、NbQPT2、NbA622、NbMYC2α及NbERF115的表达均受到显著抑制，但茉莉素诱导的NbPMT1a表达不受影响。同时，nbaidp1植株根部受茉莉素诱导的尼古丁含量也明显减少。以上结果暗示NbAIDP1可能通过结合目标基因启动子区的AT-rich元件正调控尼古丁合成相关基因的转录及最终影响尼古丁含量。此外，我们还利用CRISPR/Cas9技术制备了ntspds、ntp450、ntspds/p450三类突变体，但没有获得纯合突变体。

以上研究结果表明，AIDP1通过与NtJAZ1互作，参与茉莉素调控尼古丁的合成，是茉莉素信号通路中一个新的组分。

Alkaloids are a kind of important secondary metabolites in tobacco, which mainly includes nicotine, nornicotine, anatabine and anabasine. Nicotine is the main alkaloid in tobacco, accounting for about 95% of the total content. Transcriptional regulation of key enzyme genes in nicotine synthesis pathway, e.g. PMT and QPT, is an effective way to modify nicotine content, and these genes are regulated by jasmonate (JA) signaling pathway. The process of JA-induced nicotine formation is mainly as follows: when plants are subjected to external stress, the content of JA and its analogues increases, jasmonate ZIM domain (JAZ) protein is recruited by COI1 and degraded by 26S proteasome after ubiquitination. Subsequently, the MYC2 transcription factor was released and bound to the G-box of the PMT or QPT promoters to activate its transcription. On the other hand, MYC2 also activated the transcription of ERF transcription factors, which bind to the GCC-box of PMT or QPT promoter to activate its transcription. Moreover, MYC2 activated the transcription of JAZs, and then form a feedback inhibition loop to balance JA signaling. Previous extensive studies have shown that the key components of JA signaling pathway, such as NtCOI1 and NtJAZs, are important regulators of nicotine synthesis. Therefore, to obtain the new components of JA signaling pathway will provide important clues for further elucidating the molecular mechanism on regulating nicotine synthesis. Our prior study showed that AT-rich interaction domain containing protein 1 interacts with NtJAZ1 in yeast two-hybrid screening system. Furthermore, it was proved that NtAIDP1 binds to the AT-rich motif of GAG fragment on NtPMT1a promoter and activate its transcriptional activity. However, the detail mechanism on AIDP1 regulating nicotine formation remains elusive.

    Here, we firstly performed luciferase complementation assays in tobacco leaves, and showed that NtAIDP1 interacts with NtJAZ1, NtMYC2a and NtERF115, which is dependent on its C-terminal, but independent on its N-terminal of NtAIDP1. Moreover, the transgenic Nicotiana. tabacum plants were generated for overexpressing NtAIDP1. Then, we found that the expression levels of NtPDF1.2 and NtJAZ1 were significantly higher in NtAIDP1-overexpressed plants than that in wild type(WT) tobacco. However, there was no significant difference between the expression levels of NtPMT1a, NtQPT2 and NtMYC2a between NtAIDP1-overexpressed and wild type plants. Then, we constructed an nbaidp1 mutant with the CRISPR/Cas9 technique, and detected the effect of the knockout of NbAIDP1 on nicotine biosynthesis in dipoloid N. benthamiana. Compared with wild-type plants, the expression of JA-inducible nicotine synthesis genes, such as NbQPT2, NbJAZ1, NbA622, NbMYC2α and NbERF115, was significantly inhibited, but the expression of NbPMT1a was not affected in nbaidp1 in response to MeJA treatment. Furthermore, nicotine levels induced by JA were also significantly lower in the roots of nbaidp1 than in wide type plants. In addition, we generated ntspds (spermidine synthase)、ntp450 (demethylation oxidase of P450 family member)、ntspds/p450 mutants, and will characterize the homozygous mutant lines and monitor the nicotine content in these mutants in the future.

    In summary, our results proved that NbAIDP1, as a component of the JA signalling pathway, positively regulates nicotine biosynthesis in tobacco plants.