水生所在衣藻中建立人工miRNA基因敲降技术平台

衣藻是一种单细胞真核绿藻，它是进行光合作用、鞭毛/纤毛及生物能源等领域研究的模式生物。近年来，衣藻还用来表达抗体和疫苗，在生物医学研究方面也得到越来越多的重视。由于衣藻同源重组发生的频率低，反向遗传学的研究主要依赖于siRNA和人工miRNA技术，其中人工miRNA技术具有特异性强、效率高等优点，一直是研究的重点。但传统的人工miRNA表达载体具有构建费用高、操作繁琐、筛选困难、干扰程度有限等缺点，限制了本技术的广泛应用。

中国科学院水生生物研究所研究人员在莱茵衣藻中建立了一套利用人工miRNA敲除基因的技术平台。该技术利用衣藻内源miRNA前体做为骨架，针对叶绿体相关基因VIPP1和鞭毛基因CDPK3分别构建人工miRNA，采用8条引物退火合成人工miRNA前体的一步构建法和荧光素酶基因融合表达的方法，实现快速构建和筛选的目的。具有操作简单、成本低、筛选高效等优点。

主要实验结果如下：(1)通过一步法退火合成人工miRNA前体结构，并将荧光素酶基因和一步法合成的人工miRNA前体结构融合于PsaD启动子下游共表达。通过检测荧光素酶活性或利用Photo counting 相机直接成像，快速筛选靶基因敲降克隆。靶蛋白VIPP1可下降至野生型的22-74%。(2)人工miRNA干扰效果会随时间减弱，筛选到的阳性克隆在第一月的干扰效果最好。(3)通过在荧光素酶基因和人工miRNA前体之间融合入内源基因RBCS2的内含子1或内含子3，明显增强了荧光素酶活性和人工miRNA的干扰效果。较未加内含子的阳性克隆，荧光素酶活性分别增强至333%或401%，而靶蛋白VIPP1含量也比野生型下降至5%或3%。将内含子3融合入靶基因为CDPK3的人工miRNA载体中，也可使靶蛋白CDPK3下降至野生型的5%。(4)诱导型NIT1启动子在人工miRNA表达载体中同样适用，通过更换培养基中氮源为氨基盐或硝酸盐，从而抑制或诱导干扰载体的表达。

该研究建立的高效人工miRNA技术平台，对于推进衣藻基因功能的研究有着重要的意义，同时该方法还可用于其它的模式生物。该研究主要由水生所博士研究生胡金璐等人完成，通讯作者为黄开耀研究员和王高鸿副研究员。相关论文已在线发表于The Plant Journal杂志。

原文摘要：

Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii

Jinlu Hu, Xuan Deng, Ning Shao, Gaohong Wang andKaiyao Huang

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 plastids 1) and the flagellar protein CDPK3 (calcium-dependent protein kinase 3). Adding an intron from RBCS2 (ribulose bisphosphate carboxylase/oxygenase small subunit 2) 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.

作者：中科院