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题名: RNAi-based targeted gene knockdown in the model oleaginous microalgae Nannochloropsis oceanica
作者: Wei, Li1, 2; Xin, Yi1, 2; Wang, Qintao1, 2, 3; Yang, Juan3, 4; Hu, Hanhua4; Xu, Jian1, 2
关键词: RNAi ; oleaginous microalgae ; Nannochloropsis ; carbonic anhydrase ; bicarbonate transporter ; technical advance
刊名: PLANT JOURNAL
发表日期: 2017-03-01
DOI: 10.1111/tpj.13411
卷: 89, 期:6, 页:1236-1250
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: Microalgae are promising feedstock for renewable fuels such as biodiesel, yet development of industrial oleaginous strains has been hindered by the paucity and inefficiency of reverse genetics tools. Here we established an efficient RNAi-based targeted gene-knockdown method for Nannochloropsis spp., which are emerging model organisms for industrial microalgal oil production. The method achieved a 40-80% success rate in Nannochloropsis oceanica strain IMET1. When transcript level of one carbonic anhydrase (CA) was inhibited by 6283% via RNAi, mutant cells exhibited photosynthetic oxygen evolution (POE) rates that were 68-100% higher than wild-type (WT) at pH 6.0, equivalent to WT at pH 8.2, yet 39-45% lower than WT at pH 9.0. Moreover, the mutant POE rates were negatively correlated with the increase of culture pH, an exact opposite of WT. Thus, a dynamic carbon concentration mechanism (CCM) that is highly sensitive to pH homeostasis was revealed, where the CA inhibition likely partially abrogated the mechanism that normally deactivates CCM under a high level of dissolved CO2. Extension of the method to another sequenced N. oceanica strain of CCMP 1779 demonstrated comparable performance. Finally, McrBC-PCR followed by bisulfite sequencing revealed that the gene knockdown is mediated by the CG, CHG and CHH types of DNA methylation at the coding region of the targeted gene. The efficiency, robustness and general applicability of this reverse genetics approach suggested the possibility of large-scale RNAi-based gene function screening in industrial microalgae.
关键词[WOS]: DIATOM PHAEODACTYLUM-TRICORNUTUM ; CYANIDIOSCHYZON-MEROLAE 10D ; GENOME REVEALS ; HOMOLOGOUS RECOMBINATION ; HIGH-THROUGHPUT ; CHLAMYDOMONAS-REINHARDTII ; TRANSCRIPTOMIC ANALYSIS ; NUCLEAR TRANSFORMATION ; METHYLATION ANALYSIS ; FUNCTIONAL GENOMICS
语种: 英语
项目资助者: National Natural Science Foundation of China(31425002 ; Ministry of Science and Technology of China(2012CB721101 ; Biological Carbon Sequestration Program from Chinese Academy of Sciences(KSZD-EW-Z-017 ; Young Investigator Program from Natural Science Foundation of Shandong Province(ZR2015CQ003) ; 41576144) ; 2012AA02A707) ; ZDRW-ZS2016-3)
WOS记录号: WOS:000395811000013
ISSN号: 0960-7412
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内容类型: 期刊论文
URI标识: http://ir.ihb.ac.cn/handle/342005/28957
Appears in Collections:藻类生物学及应用研究中心_期刊论文

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作者单位: 1.Chinese Acad Sci, Qingdao Inst BioEnergy & Bioproc Technol, CAS Key Lab Biofuels, Single Cell Ctr, Qingdao 266101, Shandong, Peoples R China
2.Chinese Acad Sci, Qingdao Inst BioEnergy & Bioproc Technol, Shandong Key Lab Energy Genet, Qingdao 266101, Shandong, Peoples R China
3.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
4.Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Hubei, Peoples R China
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