Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii
Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii
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莱茵衣藻人工microRNA系统的快速构建与筛选
DOI：
10.1111/tpj.12606
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发表时间：
2014-09-01
期刊：
影响因子：
7.2
通讯作者：
Huang, Kaiyao
中科院分区：
文献类型：
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作者：
Hu, Jinlu;Deng, Xuan;Huang, Kaiyao
单细胞绿色藻类莱茵衣藻是研究鞭毛/纤毛和光合作用的经典模型，最近已被开发用于生产生物药物和生物燃料。由于同源重组的频率较低，衣原体的反向遗传操作主要依赖于基于miRNA和siRNA的敲除方法。然而，构建人工miRNA载体的困难、敲低转化体的费力筛选以及外源miRNA构建体的不期望的表观遗传沉默限制了它们的应用。我们已经建立了一个一步程序，通过退火约40个核苷酸的8个寡核苷酸构建人工miRNA前体。在最终的构建体中，Gaussia princeps荧光素酶基因（G-Luc）位于启动子和人工miRNA前体之间，以便通过使用光子计数相机观察荧光素酶发光来快速筛选敲低菌株。此外，转化体的荧光素酶活性与两种测试靶蛋白的敲低水平相关：叶绿体蛋白VIPP 1（质体中的囊泡诱导蛋白1）和鞭毛蛋白CDPK 3（钙依赖性蛋白激酶3）。将来自RBCS 2（核酮糖二磷酸羧化酶/加氧酶小亚基2）的内含子添加到miRNA构建体中增强了荧光素酶活性和miRNA敲除效率。第二个miRNA载体掺入硝酸还原酶基因的启动子以允许人工miRNA的诱导型表达。这些载体将促进人工miRNA的应用，并为研究衣原体的表观遗传学机制提供工具，也可能适用于其他模式生物。
The unicellular green algae Chlamydomonas reinhardtii is a classic model for the study of flagella/cilia and photosynthesis, and it has recently been exploited for producing biopharmaceuticals and biofuel. Due to the low frequency of homologous recombination, reverse genetic manipulation in Chlamydomonas relies mainly on miRNA- and siRNA-based knockdown methods. However, the difficulty in constructing artificial miRNA vectors, laborious screening of knockdown transformants, and undesired epigenetic silencing of exogenous miRNA constructs limit their application. We have established a one-step procedure to construct an artificial miRNA precursor by annealing eight oligonucleotides of approximately 40 nucleotides. In the final construct, the Gaussia princeps luciferase gene (G-Luc) is positioned between the promoter and the artificial miRNA precursor so that knockdown strains may quickly be screened by visualizing luciferase luminescence using a photon-counting camera. Furthermore, the luciferase activity of transformants correlates with the knockdown level of two test target proteins: the chloroplast protein VIPP1 (vesicle inducing protein in plastids1) and the flagellar protein CDPK3 (calcium-dependent protein kinase3). Adding an intron from RBCS2 (ribulose bisphosphate carboxylase/oxygenase small subunit2) to the miRNA construct enhanced both the luciferase activity and the miRNA knockdown efficiency. A second miRNA vector incorporated the promoter of the nitrate reductase gene to allow inducible expression of the artificial miRNA. These vectors will facilitate application of the artificial miRNA and provide tools for studying the mechanism of epigenetics in Chlamydomonas, and may also be adapted for use in other model organisms.