中国科学院水生生物研究所机构知识库
Advanced  
IHB OpenIR  > 藻类生物学及应用研究中心  > 期刊论文
题名: Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii
作者: Hu, Jinlu1, 2; Deng, Xuan1; Shao, Ning3; Wang, Gaohong1; Huang, Kaiyao1
关键词: RNA silencing ; miRNA ; luciferase ; epigenetics ; flagella ; technical advance
刊名: PLANT JOURNAL
发表日期: 2014-09-01
DOI: 10.1111/tpj.12606
卷: 79, 期:6, 页:1052-1064
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology ; Life Sciences & Biomedicine
类目[WOS]: Plant Sciences
研究领域[WOS]: Plant Sciences
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.
关键词[WOS]: GENE-EXPRESSION ; HOMOLOGOUS RECOMBINATION ; SMALL RNAS ; ALGA ; EVOLUTION ; MIRNAS ; TRANSFORMATION ; DEGRADATION ; TRANSGENE ; PROTEINS
语种: 英语
WOS记录号: WOS:000341515600014
ISSN号: 0960-7412
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.ihb.ac.cn/handle/342005/20400
Appears in Collections:藻类生物学及应用研究中心_期刊论文

Files in This Item: Download All
File Name/ File Size Content Type Version Access License
Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii.pdf(788KB)----开放获取View Download

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

Recommended Citation:
Hu, Jinlu; Deng, Xuan; Shao, Ning; Wang, Gaohong; Huang, Kaiyao.Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii,PLANT JOURNAL,2014,():
Service
Recommend this item
Sava as my favorate item
Show this item's statistics
Export Endnote File
Google Scholar
Similar articles in Google Scholar
[Hu, Jinlu]'s Articles
[Deng, Xuan]'s Articles
[Shao, Ning]'s Articles
CSDL cross search
Similar articles in CSDL Cross Search
[Hu, Jinlu]‘s Articles
[Deng, Xuan]‘s Articles
[Shao, Ning]‘s Articles
Related Copyright Policies
Null
Social Bookmarking
Add to CiteULike Add to Connotea Add to Del.icio.us Add to Digg Add to Reddit
文件名: Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii.pdf
格式: Adobe PDF
此文件暂不支持浏览
所有评论 (0)
暂无评论
 
评注功能仅针对注册用户开放,请您登录
您对该条目有什么异议,请填写以下表单,管理员会尽快联系您。
内 容:
Email:  *
单位:
验证码:   刷新
您在IR的使用过程中有什么好的想法或者建议可以反馈给我们。
标 题:
 *
内 容:
Email:  *
验证码:   刷新

Items in IR are protected by copyright, with all rights reserved, unless otherwise indicated.

 

 

Valid XHTML 1.0!
Copyright © 2007-2016  中国科学院水生生物研究所 - Feedback
Powered by CSpace