Division of Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
Department of Plant and Microbial Biology, University of California, Berkeley, California, United States of America.
PLoS Genet. 2021 Sep 7;17(9):e1009725. doi: 10.1371/journal.pgen.1009725. eCollection 2021 Sep.
Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4. The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas, land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis.
大规模的突变体文库对于遗传研究是不可或缺的，下一代基因组测序技术的发展极大地推进了突变体分析的工作。在这项工作中，我们对 660 个莱茵衣藻乙酸盐需求突变体的基因组进行了测序，这些突变体是先前通过线性化质粒插入诱变生成的光合作用突变体库的一部分。通过对配对末端序列中的质粒插入位点进行映射，我们从 509 个突变体中鉴定出了 554 个插入事件，其中一个末端与质粒对齐，另一个末端与染色体位置对齐。几乎所有（96%）的事件都与质粒插入位点处基因组 DNA 的缺失、重复或更复杂的重排有关，再加上与质粒插入无关的缺失，共鉴定出 1470 个受影响的基因。这些基因的功能注释在光合作用、信号转导和四吡咯合成相关的基因中富集，这与一个富含光合作用突变体的文库是一致的。对每个突变体的中断基因进行系统的手动分析，生成了一份 253 个高可信度候选光合作用基因的列表，我们通过实验验证了两个对光自养生长至关重要的基因，CrLPA3 和 CrPSBP4。候选基因清单包括来自绿藻和植物系统发育定义的保守基因集合的 53 个基因。总的来说，70 个候选基因编码的蛋白质在衣藻、陆地植物和/或蓝藻中有先前描述的光合作用功能；14 个基因编码的蛋白质先前被证明与光合作用无关。在其余的 169 个未被描述的基因中，38 个基因编码的蛋白质没有任何功能注释，这表明我们的结果将光合作用相关的功能与这些先前未知的蛋白质联系起来。这个突变体文库，结合了揭示染色体损伤分子程度的基因组序列和由此产生的更高可信度的候选基因，将有助于推进光合作用中的基因发现和蛋白质功能分析。
