2008年11月28日，北京生命科学研究所戚益军博士实验室在The Plant Journal杂志在线发表题为“Gene silencing by artificial microRNAs in Chlamydomonas”的文章。该文报道了戚益军实验室在衣藻中建立了人工miRNA介导基因沉默的技术。
衣藻是一种单细胞藻类，它是研究叶绿体，基体和鞭毛的模式生物。2007年4月，戚益军博士实验室首先在衣藻中发现了内源的miRNAs，并且证明它可以通过切割靶基因的mRNA来下调基因的表达。基于这一发现，他们在衣藻中建立了一套利用人工miRNA敲除基因的技术。他们利用衣藻内源表达的miRNA前体做为骨架，针对MAA7和RBCS1/2两个基因分别构建了人工miRNA（amiRNAs），并在衣藻中成功地表达了人工miRNA。实验结果表明，人工miRNA可以介导相应靶基因的切割且切割位点与预测位点一致，导致MAA7和RBCS1/2这两个靶基因mRNA积累减少，转基因衣藻表现出相应的表型。而后，他们进一步构建了一个二元的人工miRNA前体，它可以同时产生两个人工miRNA，并在同一个转基因株中引起两种相应的表型。在衣藻中，人工miRNA技术的建立对于推进衣藻基因功能的研究有着重要的意义，它将在研究单个基因功能和全基因组水平的筛选中得到广泛应用。
赵涛博士是该文章的第一作者，论文的其他作者还有博士研究生王伟和白雪。戚益军博士为本文的通讯作者。此项研究为科技部863计划和北京市科委资助课题，在北京生命科学研究所完成。（生物谷Bioon.com）
生物谷推荐原始出处：
The Plant Journal doi: 10.1111/j.1365-313X.2008.03758.x
Gene silencing by artificial microRNAs in Chlamydomonas
Tao Zhao 1 , Wei Wang 1,2 , Xue Bai 1,2 and Yijun Qi 1,
1 National Institute of Biological Sciences, No.7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China
2 Graduate Program in Chinese Academy of Medical Sciences and Peking union Medical College, Beijing 100730, China.
ABSTRACT
Chlamydomonas reinhardtii is a unicellular green alga. It is a model system for studying functions of chloroplast, basal body and flagella. The completion of Chlamydomonas genomic sequence makes it possible to use reverse genetic approaches in this organism. Chlamydomonas contains a set of endogenous microRNAs (miRNAs) that down-regulate their target gene expression through mRNA cleavage. Here we developed an artificial miRNA-based strategy to knock down gene expression in Chlamydomonas. Using an endogenous Chlamydomonas miRNA precursor as the backbone, we constructed two artificial miRNAs (amiRNAs) targeting the MAA7 and RBCS1/2 genes, respectively. When overexpressed, these two amiRNAs could cleave their respective targets precisely at the predicted sites, resulting in greatly decreased accumulation of MAA7 and RBCS1/2 transcripts and expected mutant phenotypes. We further showed the two amiRNAs could be produced simultaneously from a dimeric amiRNA precursor. We anticipate that the amiRNA technology developed in this study will be very useful in assessing the functions of individual genes and in genome-wide approaches.
