2008年11月28日,，北京生命科學(xué)研究所戚益軍博士實驗室在The Plant Journal雜志在線發(fā)表題為“Gene silencing by artificial microRNAs in Chlamydomonas”的文章,。該文報道了戚益軍實驗室在衣藻中建立了人工miRNA介導(dǎo)基因沉默的技術(shù)。
衣藻是一種單細(xì)胞藻類,，它是研究葉綠體,，基體和鞭毛的模式生物。2007年4月,，戚益軍博士實驗室首先在衣藻中發(fā)現(xiàn)了內(nèi)源的miRNAs,，并且證明它可以通過切割靶基因的mRNA來下調(diào)基因的表達(dá)?；谶@一發(fā)現(xiàn)，他們在衣藻中建立了一套利用人工miRNA敲除基因的技術(shù),。他們利用衣藻內(nèi)源表達(dá)的miRNA前體做為骨架,，針對MAA7和RBCS1/2兩個基因分別構(gòu)建了人工miRNA（amiRNAs），并在衣藻中成功地表達(dá)了人工miRNA,。實驗結(jié)果表明,，人工miRNA可以介導(dǎo)相應(yīng)靶基因的切割且切割位點與預(yù)測位點一致，導(dǎo)致MAA7和RBCS1/2這兩個靶基因mRNA積累減少,，轉(zhuǎn)基因衣藻表現(xiàn)出相應(yīng)的表型,。而后，他們進(jìn)一步構(gòu)建了一個二元的人工miRNA前體,，它可以同時產(chǎn)生兩個人工miRNA,，并在同一個轉(zhuǎn)基因株中引起兩種相應(yīng)的表型。在衣藻中,，人工miRNA技術(shù)的建立對于推進(jìn)衣藻基因功能的研究有著重要的意義,，它將在研究單個基因功能和全基因組水平的篩選中得到廣泛應(yīng)用。
趙濤博士是該文章的第一作者,，論文的其他作者還有博士研究生王偉和白雪,。戚益軍博士為本文的通訊作者。此項研究為科技部863計劃和北京市科委資助課題,，在北京生命科學(xué)研究所完成,。（生物谷Bioon.com）
生物谷推薦原始出處：
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.
