Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco
Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco
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DOI：
10.1007/s00425-010-1108-y
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发表时间：
2010-04-01
期刊：
影响因子：
4.3
通讯作者：
Yuan, Ling
中科院分区：
文献类型：
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作者：
Pattanaik, Sitakanta;Kong, Que;Yuan, Ling
烟草是研究bHLH-MYB转录因子（TF）复合物对植物类黄酮生物合成途径的组合调控的常用异源系统。然而，很少有人知道的内源性烟草bHLH和MYB的TF参与的途径。异源bHLH TF基因（如玉米Lc）在烟草中的异位表达导致生殖组织中花青素产生增加，表明存在与Lc样bHLH TF相互作用的生殖组织特异性MYB TF。我们从发育中的烟草花中分离了编码R2 R3 MYB TF的基因（NtAn 2）。NtAn 2与其他已知的类黄酮相关的MYB TF具有高度的序列同源性，并且主要在发育中的花中表达。NtAn 2的组成性异位表达诱导烟草和拟南芥中的全植物花青素产生。在转基因烟草和拟南芥表达NtAn 2，这两个子集的早期和晚期类黄酮途径基因的上调。在烟草中通过RNAi抑制NtAn 2导致白花表型和晚期途径基因的抑制。酵母双杂交试验表明，NtAn 2可以与五个异源bHLH TF相互作用，已知诱导其他物种，包括玉米，紫苏，金鱼草和拟南芥花色素苷的合成。使用分裂YFP的双分子荧光互补证明NtAn 2与烟草细胞中的Lc相互作用，并且该复合物定位于细胞核。NtAn 2和Lc或拟南芥TT 8在烟草原生质体中的瞬时共表达激活了两个关键类黄酮途径基因的启动子，查耳酮合成酶和二氢黄酮醇还原酶。这些结果表明NtAn 2是控制烟草生殖组织花色素苷产生的关键基因。
Tobacco is a commonly used heterologous system for studying combinatorial regulation of the flavonoid biosynthetic pathway by the bHLH-MYB transcription factor (TF) complex in plants. However, little is known about the endogenous tobacco bHLH and MYB TFs involved in the pathway. Ectopic expression in tobacco of heterologous bHLH TF genes, such as maize Lc, leads to increased anthocyanin production in the reproductive tissues, suggesting the presence of a reproductive tissue-specific MYB TF that interacts with the Lc-like bHLH TFs. We isolated a gene (NtAn2) encoding a R2R3 MYB TF from developing tobacco flowers. NtAn2 shares high sequence homology with other known flavonoid-related MYB TFs and is mostly expressed in developing flowers. Constitutive ectopic expression of NtAn2 induces whole-plant anthocyanin production in tobacco and Arabidopsis. In transgenic tobacco and Arabidopsis expressing NtAn2, both subsets of early and late flavonoid pathway genes are up-regulated. Suppression of NtAn2 by RNAi in tobacco resulted in a white-flowered phenotype and the inhibition of the late pathway genes. Yeast two-hybrid assays demonstrated that NtAn2 can interact with five heterologous bHLH TFs known to induce anthocyanin synthesis in other species including maize, perilla, snapdragon and Arabidopsis. Bimolecular fluorescent complementation using split YFP demonstrated that NtAn2 interacts with Lc in tobacco cells and that the complex is localized to nuclei. Transient co-expression of NtAn2 and Lc or Arabidopsis TT8 in tobacco protoplasts activated the promoters of two key flavonoid pathway genes, chalcone synthase and dihydroflavonol reductase. These results suggest that NtAn2 is a key gene controlling anthocyanin production in reproductive tissues of tobacco.