Department of Biology, University of York, York YO10 5DD, UK.
Department Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK.
Plant Cell. 2021 May 31;33(4):1161-1181. doi: 10.1093/plcell/koab024.
The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation. Most gene cloning is polymerase chain reaction (PCR)�or DNA synthesis-dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures. Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection. To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO2 concentrating mechanism (CCM), with an overall cloning success rate of 77%. Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kb. Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants. We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research.
克隆基因的能力极大地推动了细胞和分子生物学研究，使研究人员能够生成荧光蛋白融合物以进行定位，并通过突变互补来确认遗传因果关系。大多数基因克隆依赖于聚合酶链式反应（PCR）或 DNA 合成，随着基因大小的增加，特别是如果它们包含复杂区域，这会变得昂贵且具有技术挑战性。这一直是莱茵衣藻研究界的一个长期挑战，因为这种藻类有很高比例的基因含有复杂的序列结构。在这里，我们通过开发一种用于快速平行克隆莱茵衣藻细菌人工染色体文库中基因的重组工程管道来克服了这些挑战。为了生成用于定位的荧光蛋白融合物，我们以批处理和高通量两种规模将该管道应用于 203 个与莱茵衣藻 CO2 浓缩机制（CCM）相关的基因，总体克隆成功率为 77%。克隆成功率与基因大小和复杂性无关，克隆的基因最大可达 23 kb。对 CCM 目标的一部分进行定位证实了先前的质谱数据，确定了新的淀粉核成分，并能够对突变体进行互补。我们提供载体和详细的协议，以方便采用这项技术，我们预计这将为藻类和植物研究开辟新的可能性。
