Building a multipurpose insertional mutant library for forward and reverse genetics in Chlamydomonas.
Building a multipurpose insertional mutant library for forward and reverse genetics in Chlamydomonas.
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构建衣藻正向和反向遗传学的多用途插入突变体库
DOI：
10.1186/s13007-017-0183-5
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发表时间：
2017
期刊：
影响因子：
5.1
通讯作者：
Huang K
中科院分区：
文献类型：
--
作者：
Cheng X;Liu G;Ke W;Zhao L;Lv B;Ma X;Xu N;Xia X;Deng X;Zheng C;Huang K
单细胞绿色衣藻，莱茵衣藻，是研究鞭毛和生物燃料的经典模型。然而，精确的基因编辑，如规则间隔短回文重复序列（CRISPR）和CRISPR相关蛋白（Cas9）系统，并没有在这种生物中广泛使用。通过聚合酶链反应筛选随机插入突变体库，为获得单个基因的无效突变体提供了一种新的策略。但是建立、筛选和维护这样一个图书馆既耗时又昂贵。通过选择合适的亲本菌株，使用琼脂平板保持单个突变体，并设计用于文库筛选的插入盒特异性引物，我们成功地产生并保持了约150，000个衣原体插入突变体，其用于反向和正向遗传学分析。我们获得了26个突变体对应的20个基因，并确定了967运动缺陷突变体，其中包括10个突变体的鞭毛内运输复合物在基体的缺陷积累。我们还获得了929个缺氮后油滴组装缺陷的突变体。此外，还构建了一种新的插入盒，其两端具有剪接供体序列，这提高了基因中断的效率。总之，该文库为获得感兴趣基因的突变体和筛选具有特定表型的突变体提供了多功能平台。
The unicellular green alga, Chlamydomonas reinhardtii, is a classic model for studying flagella and biofuel. However, precise gene editing, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas9) system, is not widely used in this organism. Screening of random insertional mutant libraries by polymerase chain reaction provides an alternate strategy to obtain null mutants of individual gene. But building, screening, and maintaining such a library was time-consuming and expensive. By selecting a suitable parental strain, keeping individual mutants using the agar plate, and designing an insertion cassette-specific primer for library screening, we successfully generated and maintained ~150,000 insertional mutants of Chlamydomonas, which was used for both reverse and forward genetics analysis. We obtained 26 individual mutants corresponding to 20 genes and identified 967 motility-defect mutants including 10 mutants with defective accumulation of intraflagellar transport complex at the basal body. We also obtained 929 mutants defective in oil droplet assembly after nitrogen deprivation. Furthermore, a new insertion cassette with splicing donor sequences at both ends was also constructed, which increased the efficiency of gene interruption. In summary, this library provides a multifunctional platform both for obtaining mutants of interested genes and for screening of mutants with specific phenotype.