The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Science, Shandong University, Jinan, Shandong, China.
Maize Institute of Shandong Academy of Agricultural Sciences, Jinan, Shandong, China.
BMC Biotechnol. 2018 Sep 21;18(1):59. doi: 10.1186/s12896-018-0470-x.
Transgenic technology has become an important technique for crop genetic improvement. The application of well-characterized promoters is essential for developing a vector system for efficient genetic transformation. Therefore, isolation and functional validation of more alternative constitutive promoters to the CaMV35S promoter is highly desirable.
In this study, a 2093-bp sequence upstream of the translation initiation codon ATG of AtSCPL30 was isolated as the full-length promoter (PD1). To characterize the AtSCPL30 promoter (PD1) and eight 5' deleted fragments (PD2-PD9) of different lengths were fused with GUS to produce the promoter::GUS plasmids and were translocated into Nicotiana benthamiana. PD1-PD9 could confer strong and constitutive expression of transgenes in almost all tissues and development stages in Nicotiana benthamiana transgenic plants. Additionally, PD2-PD7 drove transgene expression consistently over twofold higher than the well-used CaMV35S promoter under normal and stress conditions. Among them, PD7 was only 456 bp in length, and its transcriptional activity was comparable to that of PD2-PD6. Moreover, GUS transient assay in the leaves of Nicotiana benthamiana revealed that the 162-bp (- 456~ - 295 bp) and 111-bp (- 294~ - 184 bp) fragments from the AtSCPL30 promoter could increase the transcriptional activity of mini35S up to 16- and 18-fold, respectively.
As a small constitutive strong promoter of plant origin, PD7 has the advantage of biosafety and reduces the probability of transgene silencing compared to the virus-derived CaMV35S promoter. PD7 would also be an alternative constitutive promoter to the CaMV35S promoter when multigene transformation was performed in the same vector, thereby avoiding the overuse of the CaMV35S promoter and allowing for the successful application of transgenic technology. And, the 162- and 111-bp fragments will also be very useful for synthetic promoter design based on their high enhancer activities.
转基因技术已成为作物遗传改良的重要手段。开发高效遗传转化载体系统的关键是使用特征明确的启动子。因此，需要分离和验证更多替代 CaMV35S 启动子的组成型启动子。
本研究从 AtSCPL30 翻译起始密码子 ATG 上游 2093bp 处分离出全长启动子（PD1）。为了对 AtSCPL30 启动子（PD1）进行功能验证，构建了 8 个长度不同的 5'缺失片段（PD2-PD9），并与 GUS 融合，生成启动子::GUS 质粒，然后转入黄花烟。PD1-PD9 可在黄花烟转基因植株的几乎所有组织和发育阶段中强烈且组成型地表达转基因。此外，PD2-PD7 在正常和胁迫条件下，驱动转基因的表达水平始终比常用的 CaMV35S 启动子高 2 倍以上。其中，PD7 只有 456bp 长，其转录活性与 PD2-PD6 相当。此外，在黄花烟叶片中的 GUS 瞬时测定显示，AtSCPL30 启动子的 162bp（-456-295bp）和 111bp（-294-184bp）片段可分别使 mini35S 的转录活性提高 16 倍和 18 倍。
作为一种来源于植物的小型组成型强启动子，PD7 具有生物安全性优势，与病毒衍生的 CaMV35S 启动子相比，降低了转基因沉默的可能性。当在同一载体中进行多基因转化时，PD7 也可替代 CaMV35S 启动子，从而避免过度使用 CaMV35S 启动子，并使转基因技术得以成功应用。而且，162bp 和 111bp 片段因其具有较高的增强子活性，对于基于合成启动子的设计也将非常有用。
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