Trichomes are specialized hair-like organs found on epidermal cells of many terrestrial plants, which protect plant from excessive transpiration and numerous abiotic and biotic stresses. However, the genetic basis and underlying mechanisms are largely unknown in Nicotiana tabacum (common tobacco), an established model system for genetic engineering and plant breeding. In present study, we identified, cloned and characterized an unknown function transcription factor NtMYB306a from tobacco cultivar K326 trichomes. Results obtained from sequence phylogenetic tree analysis showed that NtMYB306a-encoded protein belonged to S1 subgroup of the plants' R2R3-MYB transcription factors (TFs). Observation of the green fluorescent signals from NtMYB306a-GFP fusion protein construct exhibited that NtMYB306a was localized in nucleus. In yeast transactivation assays, the transformed yeast containing pGBKT7-NtMYB306a construct was able to grow on SD/-Trp-Ade+X-α-gal selection media, signifying that NtMYB306a exhibits transcriptional activation activity. Results from qRT-PCR, in-situ hybridization and GUS staining of transgenic tobacco plants revealed that NtMYB306a is primarily expressed in tobacco trichomes, especially tall glandular trichomes (TGTs) and short glandular trichomes (SGTs). RNA sequencing (RNA-seq) and qRT-PCR analysis of the NtMYB306a-overexpressing transgenic tobacco line revealed that NtMYB306a activated the expression of a set of key target genes which were associated with wax alkane biosynthesis. Gas Chromatography-Mass Spectrometry (GC-MS) exhibited that the total alkane contents and the contents of n-C28, n-C29, n-C31, and ai-C31 alkanes in leaf exudates of NtMYB306a-OE lines (OE-3, OE-13, and OE-20) were significantly greater when compared to WT. Besides, the promoter region of NtMYB306a contained numerous stress-responsive cis-acting elements, and their differential expression towards salicylic acid and cold stress treatments reflected their roles in signal transduction and cold-stress tolerance. Together, these results suggest that NtMYB306a is necessarily a positive regulator of alkane metabolism in tobacco trichomes that does not affect the number and morphology of tobacco trichomes, and that it can be used as a candidate gene for improving stress resistance and the quality of tobacco.

中文翻译：

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NtMYB306a 基因的克隆、表征和功能分析揭示了其在烟草毛状体蜡烷生物合成和胁迫耐受性中的作用。

毛状体是在许多陆生植物的表皮细胞上发现的特殊毛发状器官，可保护植物免受过度蒸腾和众多非生物和生物胁迫。然而，在烟草（普通烟草）中，遗传基础和潜在机制在很大程度上是未知的，这是一种已建立的基因工程和植物育种模型系统。在本研究中，我们从烟草栽培品种 K326 毛状体中鉴定、克隆和表征了一个未知功能的转录因子 NtMYB306a。序列系统发育树分析结果表明，NtMYB306a编码蛋白属于植物R2R3-MYB转录因子(TFs)的S1亚群。对来自 NtMYB306a-GFP 融合蛋白构建体的绿色荧光信号的观察表明 NtMYB306a 定位于细胞核中。在酵母反式激活试验中，含有 pGBKT7-NtMYB306a 构建体的转化酵母能够在 SD/-Trp-Ade+X-α-gal 选择培养基上生长，表明 NtMYB306a 表现出转录激活活性。转基因烟草植物的 qRT-PCR、原位杂交和 GUS 染色结果表明，NtMYB306a 主要在烟草毛中表达，尤其是高腺毛 (TGT) 和短腺毛 (SGT)。对过表达 NtMYB306a 的转基因烟草品系的 RNA 测序 (RNA-seq) 和 qRT-PCR 分析表明，NtMYB306a 激活了一组与蜡烷生物合成相关的关键靶基因的表达。气相色谱-质谱 (GC-MS) 表明总烷烃含量和 n-C28、n-C29、n-C31、与 WT 相比，NtMYB306a-OE 系（OE-3、OE-13 和 OE-20）叶片分泌物中的 ai-C31 烷烃显着增加。此外，NtMYB306a的启动子区域含有大量的胁迫响应顺式作用元件，它们对水杨酸和冷胁迫处理的差异表达反映了它们在信号转导和冷胁迫耐受中的作用。总之，这些结果表明，NtMYB306a 必然是烟草毛状体中烷烃代谢的正调节因子，不影响烟草毛状体的数量和形态，并且可以用作提高烟草抗逆性和质量的候选基因。NtMYB306a的启动子区域含有大量的胁迫响应顺式作用元件，它们对水杨酸和冷胁迫处理的差异表达反映了它们在信号转导和冷胁迫耐受中的作用。总之，这些结果表明，NtMYB306a 必然是烟草毛状体中烷烃代谢的正调节因子，不影响烟草毛状体的数量和形态，并且可以用作提高烟草抗逆性和质量的候选基因。NtMYB306a的启动子区域含有大量的胁迫响应顺式作用元件，它们对水杨酸和冷胁迫处理的差异表达反映了它们在信号转导和冷胁迫耐受中的作用。总之，这些结果表明，NtMYB306a 必然是烟草毛状体中烷烃代谢的正调节因子，不影响烟草毛状体的数量和形态，并且可以用作提高烟草抗逆性和质量的候选基因。