Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii

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	Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii
	Hu, Jinlu 1,2; Deng, Xuan 1; Shao, Ning 3; Wang, Gaohong 1; Huang, Kaiyao 1
刊名	PLANT JOURNAL
	2014-09-01
卷号	79 期号:6 页码:1052-1064
关键词	RNA silencing miRNA luciferase epigenetics flagella technical advance
ISSN号	0960-7412
英文摘要	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.
WOS标题词	Science & Technology ; Life Sciences & Biomedicine
类目[WOS]	Plant Sciences
研究领域[WOS]	Plant Sciences
关键词[WOS]	GENE-EXPRESSION ; HOMOLOGOUS RECOMBINATION ; SMALL RNAS ; ALGA ; EVOLUTION ; MIRNAS ; TRANSFORMATION ; DEGRADATION ; TRANSGENE ; PROTEINS
收录类别	SCI
语种	英语
WOS记录号	WOS:000341515600014
公开日期	2015-01-20
内容类型	期刊论文
源URL	[http://ir.ihb.ac.cn/handle/342005/20400]
专题	水生生物研究所_藻类生物学及应用研究中心_期刊论文
作者单位	1.Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Hubei, Peoples R China 2.Univ Chinese Acad Sci, Beijing 100039, Peoples R China 3.Chinese Acad Sci, South China Bot Garden, Guangzhou 510650, Guangdong, Peoples R China
推荐引用方式 GB/T 7714	Hu, Jinlu,Deng, Xuan,Shao, Ning,et al. Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii[J]. PLANT JOURNAL,2014,79(6):1052-1064.
APA	Hu, Jinlu,Deng, Xuan,Shao, Ning,Wang, Gaohong,&Huang, Kaiyao.(2014).Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii.PLANT JOURNAL,79(6),1052-1064.
MLA	Hu, Jinlu,et al."Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii".PLANT JOURNAL 79.6(2014):1052-1064.