De Sutter Valerie, Vanderhaeghen Rudy, Tilleman Sofie, Lammertyn Freya, Vanhoutte Isabelle, Karimi Mansour, Inzé Dirk, Goossens Alain, Hilson Pierre
Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Technologiepark 927, B-9052 Gent, Belgium.
Plant J. 2005 Dec;44(6):1065-76. doi: 10.1111/j.1365-313X.2005.02586.x.
Although sequence information and genome annotation are improving at an impressive pace, functional ontology is still non-existent or rudimentary for most genes. In this regard, transient expression assays are very valuable for identification of short functional segments in particular pathways, because they can be performed rapidly and at a scale unattainable in stably transformed tissues. Vectors were constructed and protocols developed for systematic transient assays in plant protoplasts. To enhance throughput and reproducibility, protoplast treatments were performed entirely by a liquid-handling robot in multiwell plates, including polyethylene glycol/Ca2+ cell transfection with plasmid mixtures, washes and lysis. All transcriptional readouts were measured using a dual firefly/Renilla luciferase assay, in which the former was controlled by a reporter promoter and the latter by the 35S CaMV promoter, which served as internal normalization standard. The automated protocols were suitable for transient assays in protoplasts prepared from cell cultures of Nicotiana tabacum Bright Yellow-2 and Arabidopsis thaliana. They were implemented in a screen to discover potential regulators of genes coding for key enzymes in nicotine biosynthesis. Two novel tobacco transcription factors were found, NtORC1 and NtJAP1, that positively regulate the putrescine N-methyltransferase (PMT) promoter. In addition, combinatorial tests showed that these two factors act synergistically to induce PMT transcriptional activity. The development and use of high-throughput plant transient expression assays are discussed.
尽管序列信息和基因组注释正以惊人的速度不断完善，但对于大多数基因而言，功能本体仍然不存在或很不完善。在这方面，瞬时表达分析对于鉴定特定途径中的短功能片段非常有价值，因为它们可以快速进行，且规模是稳定转化组织中无法实现的。构建了载体并开发了方案，用于在植物原生质体中进行系统的瞬时分析。为了提高通量和重现性，原生质体处理完全由多通道板中的液体处理机器人进行，包括用质粒混合物进行聚乙二醇/Ca2+细胞转染、洗涤和裂解。所有转录读数均使用双萤火虫/海肾荧光素酶测定法进行测量，其中前者由报告基因启动子控制，后者由35S CaMV启动子控制，该启动子用作内部标准化标准。这些自动化方案适用于从烟草Bright Yellow-2细胞培养物和拟南芥制备的原生质体中的瞬时分析。它们被用于一个筛选中，以发现尼古丁生物合成中关键酶编码基因的潜在调节因子。发现了两个新的烟草转录因子，NtORC1和NtJAP1，它们正向调节腐胺N-甲基转移酶（PMT）启动子。此外，组合测试表明这两个因子协同作用以诱导PMT转录活性。本文讨论了高通量植物瞬时表达分析的开发和应用。
