An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii
An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii
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DOI：
10.1105/tpc.15.00465
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发表时间：
2016-02-01
期刊：
影响因子：
11.6
通讯作者：
Jonikas, Martin C.
中科院分区：
文献类型：
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作者：
Li, Xiaobo;Zhang, Ru;Jonikas, Martin C.
绿藻莱茵衣藻是剖析光合作用真核生物生物过程的领先单细胞模型。然而，由于难以获得特定感兴趣基因的突变体，其用途受到限制。为了产生大量的定位突变体，我们开发了高通量方法，（1）通过在琼脂培养基上繁殖和低温储存，可以轻松维护数以万计的衣藻菌株，（2）识别这些集合中的诱变插入位点和物理坐标，以及（3）通过获得> 500 bp的侧翼基因组来验证突变体库中的插入位点 序列。我们使用这些方法构建了一个稳定维护的 1935 个定位突变体文库，代表了 1562 个基因的破坏。我们进一步表征了随机选择的突变体，发现 44 个插入位点中的 33 个（75%）可以通过 PCR 确认，23 个突变体中的 17 个（74%）包含单个插入。为了证明该文库阐明生物过程的能力，我们分析了编码藻类脂滴蛋白质组蛋白质的基因中被破坏的突变体的脂质含量。这项研究揭示了长链酰基辅酶A合成酶LCS2在从头合成的脂肪酸生产三酰基甘油中的核心作用。
The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.