An indexed, mapped mutant library enables reverse genetics studies of biological processes in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii is a leading single-celled model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods which: (1) Enable easy propagation on agar and cryogenic maintenance of tens of thousands of C. reinhardtii strains; (2) Identify mutant insertion sites and physical coordinates in such collections; (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 1,935 mapped mutants, representing disruptions in 1,562 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.

莱茵衣藻（Chlamydomonas reinhardtii）是研究光合真核生物生物学过程的旗舰单细胞模式生物。然而，由于难以获取目标基因的突变体，其应用潜力曾受到限制。为构建大量已定位的突变体，我们开发了高通量方法，可实现以下功能：(1) 便于在琼脂培养基上培养，并可超低温保藏数以万计的莱茵衣藻菌株；(2) 对该菌株库中的突变体插入位点及其物理坐标进行鉴定；(3) 通过获取插入位点侧翼超过500 bp的基因组序列，对突变体混合池中的插入位点进行验证。我们利用该方法构建了包含1935个已定位突变体的稳定保藏库，这些突变体分别破坏了1562个基因。我们进一步对随机选取的突变体进行了鉴定，结果显示44个插入位点中的33个（75%）可通过聚合酶链式反应（PCR）验证，23个突变体中的17个（74%）仅含单一位点插入。为验证该库在解析生物学过程中的应用价值，我们对编码藻类脂滴蛋白质组相关蛋白的基因发生突变的菌株进行了脂类含量分析。本研究揭示了长链酰基辅酶A合成酶LCS2在由从头合成脂肪酸生成三酰甘油过程中的核心作用。