pRbcS-T1 and pMALD1, two new trichome-specific promoters of Nicotiana tabacum, were identified and their strength and specificity were compared to those of previously described promoters in this species. Nicotiana tabacum has emerged as a suitable host for metabolic engineering of terpenoids and derivatives in tall glandular trichomes, which actively synthesize and secrete specialized metabolites. However, implementation of an entire biosynthetic pathway in glandular trichomes requires the identification of trichome-specific promoters to appropriately drive the expression of the transgenes needed to set up the desired pathway. In this context, RT-qPCR analysis was carried out on wild-type N. tabacum plants to compare the expression pattern and gene expression level of NtRbcS-T1 and NtMALD1, two newly identified genes expressed in glandular trichomes, with those of NtCYP71D16, NtCBTS2α, NtCPS2, and NtLTP1, which were reported in the literature to be specifically expressed in glandular trichomes. We show that NtRbcS-T1 and NtMALD1 are specifically expressed in glandular trichomes like NtCYP71D16, NtCBTS2α, and NtCPS2, while NtLTP1 is also expressed in other leaf tissues as well as in the stem. Transcriptional fusions of each of the six promoters to the GUS-VENUS reporter gene were introduced in N. tabacum by Agrobacterium-mediated transformation. Almost all transgenic lines displayed GUS activity in tall glandular trichomes, indicating that the appropriate cis regulatory elements were included in the selected promoter regions. However, unlike for the other promoters, no trichome-specific line was obtained for pNtLTP1:GUS-VENUS, in agreement with the RT-qPCR data. These data thus provide two new transcription promoters that could be used in metabolic engineering of glandular trichomes.

中文翻译：

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两个新的烟草毛状体特异性启动子的鉴定


鉴定了 pRbcS-T1 和 pMALD1，这是烟草的两个新的毛状体特异性启动子，并将它们的强度和特异性与该物种中先前描述的启动子进行了比较。烟草已成为高腺毛中萜类化合物和衍生物代谢工程的合适宿主，这些腺毛积极合成和分泌专门的代谢物。然而，在腺毛中实施整个生物合成途径需要鉴定毛状体特异性启动子，以适当驱动建立所需途径所需的转基因的表达。在这种情况下，对野生型烟草植物进行了 RT-qPCR 分析，以比较 NtRbcS-T1 和 NtMALD1（两个新鉴定的在腺毛中表达的基因）与 NtCYP71D16、NtCBTS2α 的表达模式和基因表达水平。 、NtCPS2和NtLTP1，文献报道它们在腺毛中特异性表达。我们发现 NtRbcS-T1 和 NtMALD1 在 NtCYP71D16、NtCBTS2α 和 NtCPS2 等腺毛中特异性表达，而 NtLTP1 在其他叶组织以及茎中也表达。通过农杆菌介导的转化将六个启动子中的每一个与GUS-VENUS报道基因的转录融合体引入烟草中。几乎所有转基因品系在高腺毛中都显示出 GUS 活性，表明所选启动子区域中包含适当的顺式调控元件。然而，与其他启动子不同，pNtLTP1:GUS-VENUS 没有获得毛状体特异性系，这与 RT-qPCR 数据一致。 因此，这些数据提供了两种新的转录启动子，可用于腺毛的代谢工程。