Department of Gene Function and Regulation, Institute of Life Sciences, Department of Biotechnology, Government of India, Nalco Square, Chandrasekherpur, Bhubaneswar, Orissa, India.
PLoS One. 2011;6(9):e24627. doi: 10.1371/journal.pone.0024627. Epub 2011 Sep 9.
Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy.
METHODOLOGY/PRINCIPAL FINDINGS: We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, -271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared.
We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety of plant cells.
设计具有降低序列同源性和增强启动子活性的功能高效重组启动子，对于成功地在植物细胞中堆叠或叠加基因以开发表达所需性状的转基因植物来说将是一个重要步骤。此外，关于转基因在启动子控制下在植物细胞中的特异性表达的基本知识对于评估启动子的功效至关重要。
方法/主要发现：我们构建了一组包含不同的上游激活序列（UAS）的重组启动子Mirabilis mosaic 病毒亚基因组转录物（MS8，-306 至+27）和包含 figwort mosaic 病毒亚基因组转录物启动子（FS3，-271 至+31）的 TATA 核心结构域。将与 GUS 和 GFP 报告基因偶联的重组启动子在烟草原生质体中进行了测试。在这些启动子中，369bp 长的杂种亚基因组转录物启动子（MSgt-FSgt）在瞬时和转基因系统中均表现出最高的活性。在瞬时系统中，MSgt-FSgt 相对于 CaMV35S、MS8 和 FS3 启动子分别提高了 10.31、2.86 和 2.18 倍的活性。在转基因烟草（Nicotiana tabaccum，var. Samsun NN）和拟南芥植物中，MSgt-FSgt 杂种启动子的活性分别比 CaMV35S 启动子强 14.22 和 7.16 倍。通过 qRT-PCR 鉴定了转基因烟草植物中 GUS 活性与 uidA-mRNA 水平之间的相关性。CaMV35S 和 MSgt-FSgt 启动子均导致基因沉默，但在 MSgt-FSgt 启动子的情况下，沉默程度低于 CaMV35S。使用共聚焦激光扫描显微镜对 MSgt-FSgt 和 CaMV35S 启动子驱动的单个植物细胞中的 GUS 活性进行定量，并进行比较。
我们提出了在本研究中开发的强重组启动子 MSgt-FSgt，可用于在各种植物细胞中高效组成型表达转基因。
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