PDF(Pubmed)
Abstract：
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.
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.
摘要：
转基因技术已成为作物遗传改良的重要技术。良好表征的启动子的应用对于开发用于有效遗传转化的载体系统至关重要。因此,对CaMV35S启动子的更多替代组成型启动子的分离和功能验证是非常需要的。
在这项研究中,将AtSCPL30的翻译起始密码子ATG上游的2093-bp序列分离为全长启动子(PD1)。为了表征AtSCPL30启动子（PD1）和8个不同长度的5'缺失片段（PD2-PD9）与GUS融合以产生启动子：：GUS质粒，并将其转位到Nicotianabenthamiana中。PD1-PD9可以在烟草转基因植物的几乎所有组织和发育阶段中赋予转基因的强大和组成型表达。此外,在正常和胁迫条件下，PD2-PD7驱动转基因表达始终比使用良好的CaMV35S启动子高两倍。其中,PD7长度仅为456bp，其转录活性与PD2-PD6相当。此外，烟草叶片中的GUS瞬时测定表明，AtSCPL30启动子的162bp（-456〜-295bp）和111bp（-294〜-184bp）片段可以将mini35S的转录活性提高到16-和18-倍，分别。
作为植物来源的小组成型强启动子，与病毒来源的CaMV35S启动子相比,PD7具有生物安全性的优势并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也将是CaMV35S启动子的替代组成型启动子。从而避免了CaMV35S启动子的过度使用,并允许转基因技术的成功应用。And,基于它们的高增强子活性,162-和111-bp片段对于合成启动子设计也是非常有用的。
在这项研究中,将AtSCPL30的翻译起始密码子ATG上游的2093-bp序列分离为全长启动子(PD1)。为了表征AtSCPL30启动子（PD1）和8个不同长度的5'缺失片段（PD2-PD9）与GUS融合以产生启动子：：GUS质粒，并将其转位到Nicotianabenthamiana中。PD1-PD9可以在烟草转基因植物的几乎所有组织和发育阶段中赋予转基因的强大和组成型表达。此外,在正常和胁迫条件下，PD2-PD7驱动转基因表达始终比使用良好的CaMV35S启动子高两倍。其中,PD7长度仅为456bp，其转录活性与PD2-PD6相当。此外，烟草叶片中的GUS瞬时测定表明，AtSCPL30启动子的162bp（-456〜-295bp）和111bp（-294〜-184bp）片段可以将mini35S的转录活性提高到16-和18-倍，分别。
作为植物来源的小组成型强启动子，与病毒来源的CaMV35S启动子相比,PD7具有生物安全性的优势并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也将是CaMV35S启动子的替代组成型启动子。从而避免了CaMV35S启动子的过度使用,并允许转基因技术的成功应用。And,基于它们的高增强子活性,162-和111-bp片段对于合成启动子设计也是非常有用的。
