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排序方式排序或筛选搜索：ProductsVideosKnowledgebaseSearching... 1) { page--;运行搜索（页面）； } return false;\"><上一个Page 0 of 0>NextSortFilterClose添加到购物车-+添加到购物车购买数量并节省！排序方式排序或筛选搜索：ProductsVideosKnowledgebaseSearching... 1) { page--;运行搜索（页面）； } return false;\"><上一个Page 0 of 0>NextSortFilterClose添加到购物车-+添加到购物车购买数量并节省！The Plant JournalVolume 89, Issue 6 p. 1236-1250 Technical Advance Free Access RNAi-based targeted gene knockdown in the model oleaginous microalgae Nannochloropsis oceanica Li Wei, Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 ChinaSearch for more papers by this authorYi Xin, Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 ChinaSearch for more papers by this authorQintao Wang, Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China University of Chinese Academy of Sciences, Beijing, 100049 ChinaSearch for more papers by this authorJuan Yang, University of Chinese Academy of Sciences, Beijing, 100049 China Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072 ChinaSearch for more papers by this authorHanhua Hu, Corresponding Author hanhuahu@ihb.ac.cn Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072 ChinaFor correspondence (e-mails xujian@qibebt.ac.cn or hanhuahu@ihb.ac.cn).Search for more papers by this authorJian Xu, Corresponding Author xujian@qibebt.ac.cn Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 ChinaFor correspondence (e-mails xujian@qibebt.ac.cn or hanhuahu@ihb.ac.cn).Search for more papers by this author Li Wei, Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 ChinaSearch for more papers by this authorYi Xin, Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 ChinaSearch for more papers by this authorQintao Wang, Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 China University of Chinese Academy of Sciences, Beijing, 100049 ChinaSearch for more papers by this authorJuan Yang, University of Chinese Academy of Sciences, Beijing, 100049 China Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072 ChinaSearch for more papers by this authorHanhua Hu, Corresponding Author hanhuahu@ihb.ac.cn Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072 ChinaFor correspondence (e-mails xujian@qibebt.ac.cn or hanhuahu@ihb.ac.cn).Search for more papers by this authorJian Xu, Corresponding Author xujian@qibebt.ac.cn Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101 ChinaFor correspondence (e-mails xujian@qibebt.ac.cn or hanhuahu@ihb.ac.cn).Search for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Summary 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 62–83% 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. Introduction Microalgae are a large group of photoautotrophic eukaryotic organisms that play important roles in marine, freshwater and even terrestrial ecosystems on earth (Zhu etal., 2013; Piggott etal., 2015). For example, marine microalgae are among the most significant contributors of biological fixation and cycling of atmospheric CO2 (Riebesell etal., 2009). Moreover, many microalgae found in nature are oleaginous, i.e. they are capable of storing solar energy and carbon dioxide in the form of triacylglycerol (TAG) under certain environmental conditions (Hu etal., 2008). Thus, there has been growing interest worldwide in selection and development of robust oleaginous microalgae as a potential source of biomass feedstock for biofuels and biomaterials, and moreover for reducing CO2 emissions (Wijffels and Barbosa, 2010; Georgianna and Mayfield, 2012; Zaimes and Khanna, 2013). Rational development of industrial strains to fulfill such potential of microalgae generally requires both genome-wide knowledge of cellular metabolism and the capability to engineer them. However, although the number of sequenced genomes of oleaginous microalgae has been rapidly growing (Armbrust etal., 2004; Matsuzaki etal., 2004; Derelle etal., 2006; Merchant etal., 2007; Bowler etal., 2008; Worden etal., 2009; Blanc etal., 2010; Cock etal., 2010; Prochnik etal., 2010; Gobler etal., 2011; Radakovits etal., 2012; Vieler etal., 2012; Corteggiani Carpinelli etal., 2014; Wang etal., 2014a; Fan etal., 2015), the toolbox of reverse genetics for oleaginous microalgae has been limited and inefficient. For example, insertional mutagenesis and targeted gene disruption have been reported in a few algae, such as Chlamydomonas reinhardtii (Sodeinde and Kindle, 1993; Dent etal., 2005), Phaeodactylum tricornutum (Daboussi etal., 2014), Cyanidioschyzon merolae (Minoda etal., 2004) and Nannochloropsis spp. (Kilian etal., 2011; Perin etal., 2015). However, these tools can be laborious, difficult to replicate in other strains, limited by significant biological constraints, or require extensive screening. As a result, such tools are not yet widely available to the community. On the other hand, generation of loss-of-function mutants by random insertional or chemical mutagenesis, despite being an excellent strategy to screen for new loci underlying a particular function, is unable to probe gene function in a direct and targeted manner; moreover, diploid organisms with unknown sexual cycles can be difficult to tackle using this approach (Parker etal., 2008). These limitations have greatly hindered hypothesis-driven testing of gene and pathway functions, and impeded rational strain development in oleaginous microalgae (Hlavova etal., 2015). RNA-mediated silencing is a promising tool for targeted gene knockdown in algae that harbor functional RNAi machinery. RNA interference (RNAi) is a post-transcriptional process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules (Carthew and Sontheimer, 2009). RNAi has been proven to be efficient in interfering with gene expression in a wide range of organisms from bacteria to animals and plants (Hammond etal., 2001; Watanabe, 2011). In microalgae, RNAi as a reverse genetics tool was established for only a few species, such as C.reinhardtii (Rohr etal., 2004), P.tricornutum (De Riso etal., 2009), Penium margaritaceum (Sorensen etal., 2014) and Dunaliella salina (Jia etal., 2009). However, RNAi machineries and their feasibility as reverse genetics tools remain largely unexplored in oleaginous microalgae. Nannochloropsis spp. are a group of unicellular photosynthetic heterokonts distributed widely in sea, fresh and brackish waters. As Eustigmatophyceae, they are phylogenetically more closely related to diatoms than to green algae. They have been chosen world-wide as model organisms for industry-scale oil production with microalgae due to their ability to grow rapidly, synthesize large amounts of TAGs and high-value polyunsaturated fatty acids (e.g. eicosapentaenoic acid), and tolerate broad environmental and culture conditions (Wang etal., 2012). The genomes of at least eight Nannochloropsis strains from six species have been sequenced (Pan etal., 2011; Radakovits etal., 2012; Vieler etal., 2012; Wei etal., 2013; Corteggiani Carpinelli etal., 2014; Wang etal., 2014a). Moreover, both condition-specific and time-series datasets of transcriptomes have been produced (Li etal., 2014b; Jia etal., 2015; Poliner etal., 2015). These rich resources of gene expression data together provide a foundation for identification and dissection of RNAi machineries as well as theirpotential targets, and for exploring the feasibilityofexploiting RNAi as reverse genetics tools in Nannochloropsis spp. Here, by employing two strains of industrial oleaginous microalga N.oceanica as model, we established an RNAi-based targeted gene-knockdown method using constructs harboring inverted repeat sequences of selected target genes. The method achieved a 40–80% success rate in N.oceanica strain IMET1, as evidenced from generation of carbonic anhydrase (CA) and bicarbonate transporter (BCT) knockdown mutants. When the transcript of one of the five CAs was reduced by 62–83%, the cells exhibited photosynthetic oxygen evolution (POE) rates that were 68–100% higher than wild-type (WT) under pH 6.0, an equivalent level to WT under pH 8.2, yet 39–45% lower than WT under pH 9.0. Moreover, the mutant POE rates are negatively correlated with the increase of culture pH, which is the exact opposite of WT. These results revealed a crucial role of this CA in carbon concentration mechanisms (CCM). Extension of the method to another sequenced N.oceanica strain of CCMP 1779 demonstrated comparable performance. We further showed 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 pave the way for large-scale RNAi-based gene function screening in the industrial microalgae. To probe the presence of RNAi machineries in Nannochloropsis spp., we identified polypeptides of three key components of Argonaute (AGO-Piwi), Dicer and RNA-dependent RNA polymerase (RDR) via BLAST searches in a database of protein and translated genomic DNA from sequenced microalgal genomes (Table1). Nannochloropsis oceanica strain IMET1 and Nannochloropsis gaditana strain CCMP526 each harbor one Dicer, two AGO-Piwi and one RDR (Table1). In these Nannochloropsis proteins, typical conserved domains were all present (Figure1a), for example the DEAD, Heli_C, dsRNA_bind, RNase domains in Dicer, the highly conserved PAZ and PiWi domains in AGO-Piwi, and the RdRP domain in RDR. PAZ is a nucleic acid-binding domain with strong preference for single-stranded nucleic acids (RNA or DNA) or RNA duplexes with single-stranded 3′ overhangs. Piwi domain is involved in dsRNA-guided hydrolysis of ssRNA and Dicer binding (Dillon etal., 2005). Moreover, each of AGO-Piwi, Dicer and RDR is highly similar in protein sequence between N.oceanica IMET1 and N.gaditana CCMP526 (76%, 71% and 45% identity, respectively). Thus, the RNAi machinery appears to be present and highly conserved in Nannochloropsis spp. ‘●’: presence of gene (number of circles indicates copy number); ‘○’: absence of gene. Organisms are ordered by phylogenetic classification, with green algae listed first. Features of the three key components in RNAi machinery in Nannochloropsis oceanica IMET1. (a) Protein domain structure of the key components of RNAi machinery. (b) Phylogenetic tree of the three core components of RNAi machinery Argonaute (AGO), Dicer, and RNA-dependent RNA polymerase (RDR). (c) Expression dynamics of the key components under N+ (nitrogen-replete) and N− (nitrogen-depleted) conditions. [Colour figure can be viewed at wileyonlinelibrary.com]. Phylogenetic analyses of the microalgal RNAi core components with Arabidopsis thaliana as outgroup revealed that the Nannochloropsis AGO-Piwi, Dicer and RDR are most closely related to those from the diatom Thalassiosira pseudonana (81% identity), the red algae Chondrus crispus (44% identity) and the green algae Klebsormidium flaccidum (79% identity; Figure1b). However, in general, sequence-based phylogenies of the three genes are distinct from one another, with each bearing little similarity to organismal phylogeny, suggesting a high degree of divergence in origin and evolution. The time-series transcriptome datasets that we have previously produced under both nitrogen-replete (N+) and nitrogen-depletion (N−) conditions for N.oceanica IMET1 revealed that AGO-Piwi (g4419), Dicer (g6768) and RDR (g2555) were all transcribed at significant levels under each condition (Figure1c; Li etal., 2014b). The AGO-Piwi transcript was largely temporally stable at a basal level under N+, yet it underwent an increase until reaching plateau at 24h under N−. Thus, expression of AGO-Piwi was sensitive to the nitrogen level. Interestingly, under both N+ and N−, both Dicer and RDR were transcriptionally upregulated until peaking at 12h, and then dropped to basal level at 24h, suggesting their expression was sensitive to growth stage yet insensitive to nitrogen-depletion stress. Overall, the genomic and transcriptomic evidence suggest that functional RNAi machinery is likely in operation in this alga. To explore the feasibility of the endogenous RNAi machinery as a reverse genetic tool, we started with knockdown of a predicted γ-type CA (g2209; FigureS1; TableS1) and a putative BCT (g1855) in N.oceanica IMET1, using inverted-repeat mediated RNAi approach. Both CA and BCT are major and key components of CCM (Raven etal., 2012), and the particular IMET1 genes were identified based on their amino acid sequence homology (Wang etal., 2014a). Under both N+ and N−, the CA and BCT were both transcriptionally downregulated first until bottoming at 12h, and then gradually recover at 24h and beyond (FigureS2a and b; Li etal., 2014b), suggesting their expression was sensitive to growth state. Nitrogen depletion inhibited the CA expression, with the strongest inhibition taking place at 12 h (FigureS2a). Yet the BCT appeared not particularly responsive to nitrogen-depletion stress (FigureS2b; Li etal., 2014b). To construct the RNAi expression vector for N.oceanica IMET1, an approximately 200-bp ‘short’ fragment (SF) and an approximately 400-bp ‘long’ fragment (LF) were selected from the targeted coding sequence so as to formulate the inhibitory RNA (primer sequences listed in TableS2). The two fragments share the first ~200bp to ensure formation of the stem-loop structure via the inverted repeat and then recognition by the Dicer enzyme (Figure2a). The two fragments were respectively amplified from a N.oceanica IMET1 cDNA library, and then ligated in sense and antisense orientations, respectively, and in tandem to the linearized diatom phir-PtGUS vector (De Riso etal., 2009) to create phir-PtNo plasmid. To drive robust gene expression, the endogenous β-tubulin promoter of N.oceanica IMET1, which is constitutively expressed under both N+ and N− conditions (Li etal., 2014b), was amplified from genomic DNA and inserted upstream of the selection marker and the inhibitory RNA, resulting in the phir-NoCA or phir-NoBCT plasmid (Figure2a; DataS1). Generation and screening of RNAi-based targeted gene knockdown mutant strains. (a) Expression cassettes containing inverted repeat of the carbonic anhydrase (CA)(g2209) gene for vector construction. LF, long fragment; SF, short fragment. Pbtub: β-tublin; bleR: Zeocin resistance gene; FcpA3′: terminator of fucoxanthin chlorophyll protein gene from Phaeodactylum tricornutum. (b) Validation of transgenic Nannochloropsis oceanica IMET1 lines by polymerase chain reaction (PCR). The lines were cultured in liquid f/2 medium supplemented with zeocin for selection based on the marker gene. The expected 436-bp band was present in the transgenic lines and absent in wild-type (WT). (c) Screening of CA knockdown lines by quantitative (q)PCR. The transcript level of the CA in IMET1 transformants under air-level CO2 was determined by real-time qPCR, and calculated from Ct values using the 2−▵▵t method after all results were normalized against the β-actin housekeeping gene. Values and error bars represent, respectively, the mean and standard deviation from three biological replicates. [Colour figure can be viewed at wileyonlinelibrary.com]. To introduce the RNAi expression vector into WT N.oceanica IMET1, the 2.3-kb-long cassette containing tub promoter, selection marker gene (ble; bleomycin resistance protein) and inhibitory RNA was polymerase chain reaction (PCR)-amplified first, as linearization of vector might improve the efficiency of electroporation (Li etal., 2014a). The PCR fragment was then transformed into IMET1 by electroporation. Surviving transformed microalgae cells were selected and subjected to PCR screening using primers targeting ble. Gel electrophoresis revealed that an expected 0.4-kb band was present in the transgenic lines yet absent in the WT (Figure2b), indicating successful delivery of the vector into IMET1 cells. To detect whether the targeted gene was silenced in the transgenic lines, transcript abundance of CA(g2209) was determined by quantitative (q)PCR for the stationary phase from cells cultivated under air-level (0.04% v/v) CO2, for each of the transgenic lines (Experimental procedures). Among the five transgenic lines randomly chosen from one plate, two of the five mutant lines, M4 and M5, exhibited a significant decrease in g2209 transcript level (inhibited by 65 and 52%, respectively) under air-level CO2, indicating successful RNAi-mediated knockdown (Figure2c). On the other hand, the transcript level of the other four CA genes in the two mutants remained largely equivalent to that in WT, suggesting high specificity of the target gene knockdown (FigureS3). Carbonic anhydrase as a key component of CCM (induced by air-level CO2) might play a crucial role in photosynthetic carbon assimilation in algae (Raven etal., 2012). To probe the consequence of the CA(g2209) knockdown, biomass accumulation rates were first compared between the mutants (M4 and M5) and WT under air-level CO2. Spot tests suggested that the growth rate of the mutants markedly decreased (Figure3a). Growth curves further revealed that the mutants, largely similar in growth rate, were out-grown by WT starting from Day 2, eventually resulting in a 20% lower OD750 than WT at Day 8 under air-level CO2 (pH of medium: 8.2–9.0; Figure3b). Growth parameters and photosynthetic activity of carbonic anhydrase (CA)-knockdown Nannochloropsis oceanica IMET1 strains. (a) Spot test and growth phenotype of CA(g2209)-knockdown lines under air-level CO2 (pH of medium: 8.2–9.0). (b) Growth curves of CA(g2209)-knockdown lines under air-level CO2 (pH of medium: 8.2–9.0). (c) Photosynthetic activity of the transgenic and wild-type (WT) lines under air-level CO2. The photosynthetic activity of photosystem II was measured at pH 8.2 using imaging_PAM during the logarithmic phase of the growth curve. (d) Biomass productivity of the CA(g2209)-knockdown lines cultured under air-level (pH of medium: 8.2–9.0) and 5% CO2 (pH of medium: 6.0–6.5). The dry weight of cells was measured after cultivation for 10days. Values and error bars represent the mean and standard deviation from three biological replicates, respectively. To examine photosynthetic activity in the mutants, logarithmic-phase cells under air-level CO2 were compared using Fv/Fm (via Imaging-PAM), which as ratio of variable fluorescence to maximal fluorescence reflects the optimal/maximal photochemical efficiency of PS II in the dark (Baker, 2008). In the CA-knockdown mutants, a decrease of approximately 25% (P 0.05) in Fv/Fm as compared with WT was observed under air-level CO2 (pH of medium: 8.2–9.0; Figure3c), suggesting reduction of photosynthetic activity. To probe whether these observed phenotypes represent a sustained long-term effect, the dry weight of algal cells was measured after 10days cultivation (Figure3d). The CA(g2209) knockdown mutants showed a 12–16% decrease in dry weight as compared with that of WT under air-level CO2 (pH of medium: 8.2–9.0), which is consistent with their distinct growth curves. However, under 5% CO2 (pH of medium: 6.0–6.5), no significant difference in either growth curve or dry weight was observed between the mutants and WT. On the other hand, it is notable that biomass accumulation rate of the WT under 5% CO2 was 15% lower than that under air-level CO2 (Figure3d). As it was not practical to maintain a fixed pH during long-term cultivations (i.e. 10days), such reduction of biomass accumulation was probably due to the acidification of culture resulting from the CO2 augmentation; for example, the optimal CO2 level for Nannochloropsis oculata NCTU-3 growth is 2% (Chiu etal., 2009). The rate of POE is a key measure of photosynthetic rate (Meunier and Popovic, 1988). POE rates in the CA mutants and WT from air-level CO2 culture were each measured under various pH values using a Clark-type oxygen electrode at a light intensity of 300μmolphotonsm−2sec−1 and at 25°C (Figure4a; Experimental procedures). In addition, g2209 transcript abundance was measured using real-time qPCR at each of the pH values, which validated suppression of the transcript in the mutants at each of the pH values as compared with WT (FigureS4). At pH 9.0, the mutants exhibited POE rates that were 39–45% lower than WT (mutants: 96 and 106μmol O2mg−1 Chlah−1, respectively; WT: 174μmol O2mg−1Chla h−1). However, at the acidic pH of 6.0, the POE rates became 68–100% higher than WT (153–180 and 105μmol O2mg−1Chla h−1, respectively). At pH 7.8, the rates were identical between the mutants and WT (both at approximately 140μmol O2mg−1 Chla h−1). In fact, in WT, the POE rate is positively correlated with the increase of pH, yet the CA mutants exhibit the exact opposite, i.e. increase of pH greatly inhibits the POE rate. Correlation of photosynthetic oxygen evolution (POE) rate with pH in wild-type (WT) and carbonic anhydrase (CA)-knockdown mutant lines of Nannochloropsis oceanica IMET1. The POE rates were measured under a variety of pH buffers using microalgal cells cultivated under air-level CO2 (0.04% v/v) (a) or 5% CO2 (b). Values and error bars represent, respectively, the mean and standard deviation from three biological replicates. [Colour figure can be viewed at wileyonlinelibrary.com]. On the other hand, when cultured under 5% CO2, at pH 9.0, the mutants display POE rates that are 20–25% lower than WT (mutants: 136 and 126μmol O2mg−1 Chlah−1, respectively; WT: 169μmol O2mg−1 Chla h−1; Figure4b), which is similar to the observation under 0.04% CO2. However, at pH 6.0, the POE rates of the mutants (165 and 175μmol O2mg−1 Chlah−1, respectively) are significantly higher than those at pH 9.0, as was the case under 0.04% CO2. Thus, under 5% CO2, the negative correlation is also apparent between POE rate and the increase of pH in the CA(g2209) mutants. How oleaginous microalgae tolerate CO2 level and the correlated pH level is poorly understood (Solovchenko and Khozin-Goldberg, 2013). Our observations here suggested a dynamic CCM that is highly sensitive to pH homeostasis. CCM generally shows a higher activity when the available dissolved inorganic carbon (DIC; CO2, CO32− and HCO3−) is limited (Raven etal., 2012); thus in WT, increase of pH leads to reduction of DIC (dissolved CO2) in medium and activates CCM to maintain or elevate the higher photosynthesis rate. Under high pH, inhibition of the CA transcript presumably compromises the proper activation or normal function of CCM, which results in lower photosynthesis than WT. However, under acidic pH, we speculate that inhibition of the CA transcript could have lifted the negative regulation that normally deactivates CCM under the exceedingly higher dissolved CO2. For example, it is possible that under acidic pH, while photosynthesis in WT was inhibited due to decrease of intracellular pH, the CA inhibition in mutants actually helps to maintain a more stable pH inside the cell, and thus contributes to the higher photosynthesis rate than in WT. To further evaluate the efficacy of the RNAi mutant generation process, a second gene, g1855, was chosen as the target for knockdown in IMET1. The gene was annotated as a BCT and thus might be involved in photosynthetic carbon fixation, like the CA. The RNAi vector was designed in a similar manner, by substituting the two inverted repeats of NoCA with those of NoBCT (DataS2). The surviving transformed microalgae cells were selected and subjected to PCR screening using primers for ble. An expected 0.4-kb band was present in the transgenic lines, while absent in WT (Figure5a), indicating success in transformation. Among five randomly selected transformants, four of them showed a significant decrease in BCT transcription as compared with that of WT (Figure5b), including the two transgenic lines of BR3 and BR4 in which the degree of inhibition reaches 74 and 56%, respectively. Thus, a success rate in generating valid RNAi knockdown mutants of up to 80% can be achieved. Characterization of the bicarbonate transporter (BCT) knockdown lines of Nannochloropsis oceanica IMET1. (a) Validation of transgenic lines by polymerase chain reaction (PCR). The lines were cultured in liquid f/2 medium supplemented with zeocin for selecting the marker gene shble. The expected 436-bp band was present in the transgenic lines and absent in wild-type (WT). (b) Transcript levels of BCT in the transgenic lines determined by real-time quantitative (q)PCR. The relative expression level of the BCT gene was calculated from Ct values using the 2−∆∆t method after all results were normalized against the β-actin housekeeping gene. Spot test (c) and growth curves (d) of two of the BCT-knockdown lines (BR3 and BR4) were shown under air-level CO2 (pH of the medium: 8.2–9.0). Values and error bars represent the mean and standard deviation from three biological replicates, respectively. Despite the severe inhibition of BCT transcripts, phenotypes of the BCT-knockdown mutants were not as prominent as those caused by CA-knockdown. Neither spot tests nor growth curves tracked by OD750 revealed a significant difference in biomass accumulation rate or growth between the BCT-knockdown lines and WT under air-level CO2 (pH of medium: 8.2–9.0; Figure5c and d). These observations, plus the insensitivity of BCT transcript to temporal change (i.e. a low transcript level of FPKM of 20–35; FPKM, fragments per kilobase of exon per million fragments mapped) during the first 48h in either N+ or N− and to culture-condition change (N− versus N+) in WT (FigureS1), suggested a constitutive and likely functionally insignificant role of BCT in carbon assimilation upon nitrogen-deficiency stress. In contrast, the lower growth rate and reduced POE of the CA(g2209)-knockdown mutant in respect to WT under air-level CO2 condition, plus the observation of consistent CA transcription at a moderate level (FPKM: 80–160) under N+ and N− (the first 48 h in either N+ or N−) and reduction in transcript level (N− versus N+; 30% lower) based on absolute abundance in WT (FigureS2), indicated that the CA might have a crucial effect on carbon assimilation, especially in response to different stresses. Together these observations provide initial functional evidence for the genes that underlie carbon assimilation in Nannochloropsis spp. To assess the stability of target gene silencing in the RNAi-based knockdown strains, the transcript level of g2209 in the CA(g2209)-knockdown lines of M4 and M5 and that of g1855 in the BCT(g1855)-knockdown lines of BR3 and BR4 under air-level CO2, after approximately 18 cycles of culture (20 days for each cycle) that span 12 months, was interrogated (FigureS5). The result suggested that the degree of inhibition remained at 54–68%, which corresponded to, respectively, 91 and 96% of those measured at the first generations of the mutant lines. Therefore, the RNAi-based suppression of target transcripts appears to be rather stable. The efficiency of such genetic approaches in microalgae can be strain specific, which can greatly hinder their actual value to the research community. To test the general applicability of our method, a second Nannochloropsis strain was selected as the chassis. Nannochloropsis oceanica CCMP1779 is another publicly available strain with genome and transcriptomic sequences available (Vieler etal., 2012; Poliner etal., 2015). A β-type CA of CCMP177911263 in CCMP1779 was selected as a target for the method demonstration (FigureS1; TableS1). The gene shares 99% amino acid sequence identity with the CA(g2018) from IMET1 and is actually more similar to bacterial CAs (42% identity to a CA from Clostridium) than to other non-Nannochloropsis microalgal CAs. The counterpart of CCMP177911263 in IMET1, g2018, showed a twofold transcriptional upregulation at 12, 24 and 48h under N− versus N+, suggesting its potential role in stress response to nitrogen availability or in oil production. Thus, this β-type CA was chosen for demonstration of gene knockdown in CCMP1779. In the expression vector, the two fragments (sense and antisense sequences) of CCMP177911263 were reversely inserted downstream of the CCMP1779 tubulin promoter (DataS3). The transformed microalgae cells were selected and subjected to PCR screening with primers for ble. A significant decrease in abundance of targeted transcript, i.e. by 60–80%, was detected in the CCMP1779 mutants, suggesting a comparable level of performance of the RNAi-based knockdown method between the two host strains (Figure6a). Characterization of a carbonic anhydrase (CA) knockdown line in Nannochloropsis oceanica CCMP 1779. (a) Transcript levels of CCMP177911263 in the transgenic lines determined by real-time quantitative polymerase chain reaction (qPCR) under low CO2. The relative expression level of the gene was calculated from Ct values using the 2−▵▵t method after all results were normalized against the β-actin housekeeping gene. Growth curves of the CCMP177911263-knockdown N.oceanica CCMP1779 lines under high-CO2 (b) and air-level CO2 (c) were shown. Values and error bars represent, respectively, the mean and standard deviation from three biological replicates. [Colour figure can be viewed at wileyonlinelibrary.com]. Interestingly, despite the absence of a detectable difference in growth phenotype under air-level (~0.04%) CO2, the two CCMP1779|11263-knockdown lines of N8 and N10 exhibited an increased cell density (with an increase of 40%) compared with WT under 5%-CO2 culture condition (Figure6b and c). Thus, in CCMP1779, CCMP177911263 is likely implicated in regulation of CCM in response to variation of CO2 concentration. Considering the identification of five CAs in both IMET1 and CCMP1779 (TableS1), the remarkable yet distinct phenotypes observed in the IMET1 CA(g2209)-knockdown mutants and the CCMP177911263-knockdown mutants suggested the likely presence in Nannochloropsis spp. of a diversified, versatile and intricate functional network of CAs. In plants, the transcriptional gene silencing triggered by small RNAs is generally associated with DNA modification (Zilberman etal., 2003; Chan, 2008). To probe whether the gene silencing in Nannochloropsis spp. is due to DNA methylation, methylation profiles of the targeted gene in the CA(g2209)-RNAi lines of N.oceanica (the upstream called MCR1, the gene region called MCR2 and MCR3, and the downstream called MCR4; Figure7a) in the CA(g2209)-knockdown lines of IMET1 (M4 and M5) were profiled using McrBC-PCR (MCR; Experimental procedures). The MCR1 and MCR4 regions but not the MCR2 and MCR3 regions were PCR-amplified from McrBC-treated genomic DNA of M4 and M5 (Figure7a), which indicated that MCR2 and MCR3 were methylated. Cytosine methylation pattern of the coding region of targeted gene carbonic anydrase (CA)(g2209) and its upstream and downstream in the gene-knockdown mutants of Nannochloropsis oceanica IMET1. (a) Polymerase chain reaction (PCR) amplifications performed on genomic DNA digested with McrBC, in the presence (+) and absence (−) of GTP, using primer sets specific for the coding region (both introns and exons) of the targeted gene and its upstream and downstream regions. (b) Methylation analysis was performed on the M4 mutant by bisulfite sequencing. Schematic representation of the methylation profile [including sense and antisense sequences of the coding region and the upstream and downstream of CA(g2209)] was provided. Only those reliable methylated sites, defined as those for which percentage of methylation all reached 100% among the 10 randomly selected PCR clones from each of the 12 amplified regions (S1–S6, plus A1–A6), were shown on the figure. The methylation events were classified into three types: CG, CHG or CHH. P, potential methylation sites; BS, methylation sites identified by bisulfite sequencing. Moreover, the methylation profiles of the targeted gene were analyzed by bisulfite sequencing (primers were listed in TableS3). The results revealed a number of methylation events in the CA(g2209) coding region (including both exons and introns) of the mutant lines but not at its upstream or downstream (Figure7b), which is consistent with the above-mentioned MCR results. Interestingly, three distinct methylation modes (CG, CHG and CHH) are present. On the sense strand (the S3, S4 and S5 regions; Figure7b), a total of 24 methylation sites (including 15 CG, two CHG and seven CHH) were found. On the antisense strand (A3, A4 and A5 regions), there were 27 methylation sites (including 11 CG, three CHG and 13 CHH). These methylated sites are considered as reliable (i.e. those shown at Figure7b), as for each of them the percentages of methylation all reached 100% among the 10 randomly selected PCR clones from each of the 12 amplified regions (S1–S6, plus A1–A6). Thus the RNAi-based gene knockdown in N.oceanica is underpinned by DNA methylation at the coding sequence of the targeted gene, including both introns and exons. This mechanistic model of RNAi-based gene knockdown appears to be largely conserved in heterokonts, as in the diatom P.tricornutum it was also reported that introduction of the RNAi-based silencing vector caused de novo DNA methylation of the coding sequence but not the promoter or terminator regions of the targeted gene (De Riso etal., 2009). It is notable, however, that in P.tricornutum only the CG type of methylation was found yet, in N.oceanica, all three types of methylation including CG, CHG and CHH were present. In parallel with rapid accumulation of oleaginous microalgae genomes, transcriptomic studies that dissect various aspects of microalgal oleaginousness have been dramatically expanding in scope (Guarnieri etal., 2011; Rismani-Yazdi etal., 2012; Dienst etal., 2014; Gwak etal., 2014; Abida etal., 2015; Hovde etal., 2015; Tanaka etal., 2015; Yao etal., 2015; Matthijs etal., 2016; Ota etal., 2016). However, the danger of using gene sequence or expression data alone to infer gene function in the absence of reverse genetics approaches can never be underestimated (Rossi etal., 2015). Furthermore, the challenge of hypothesis-driven perturbation and rational engineering of genes, pathways and networks in oleaginous microalgae remained enormous. Part of the reason is the paucity and inefficiency of current reverse genetics toolbox available for these organisms. In this work, we established RNAi-based gene silencing as an efficient reverse genetics approach in Nannochloropsis spp., which are an emerging model organism for industrial microalgal production of oil worldwide. By knocking down multiple CO2-assimilation genes that include CA and BCT and unveiling mutant phenotypes, the method demonstrates a 40–80% possibility of successful mutant generation, and up to 79% transcript level suppression in N.oceanica strain IMET1. Moreover, the method was validated using a second strain of N.oceanica, CCMP 1779. As a gene ‘knockdown’ approach, RNAi offered a number of potential advantages over gene disruption mediated by random insertion, homologous recombination or Cas9-based genome editing (Shalem etal., 2015). For example, using gene disruption, it remains difficult to evaluate the functional landscape of essential genes (i.e. gene ‘knockout’), which however can be probed via RNAi-based knockdown. Moreover, the use of RNAi in perturbing gene function and cellular network can offer a higher degree of flexibility and nimbleness than gene disruption, as RNAi targets transcripts (instead of the genome) that are in nature more volatile, diverse and sensitive to environment. Therefore, the RNAi method reported here is a valuable addition to the existing genetic toolbox of Nannochloropsis spp., which includes random insertional mutagenesis (Perin etal., 2015), homologous recombination (Kilian etal., 2011), gene overexpression (Kang etal., 2015) and Cas9-based genome editing (Wang etal., 2016a). In N.oceanica IMET1 and CCMP1779, recent studies that interrogated cellular mechanisms of TAG synthesis (Li etal., 2014b), carbon partitioning (Jia etal., 2015; Poliner etal., 2015), sterol metabolism (Lu etal., 2014b), phytohormone function (Lu etal., 2014a; Lu and Xu, 2015), stress response (Xiao etal., 2015) or genome-wide regulation by transcriptional factors (Hu etal., 2014) have yielded numerous valuable hypotheses, yet few have been validated using genetic approaches. Such a situation is expected to change rapidly as the RNAi method established here can directly contribute to testing of these and additional hypotheses. On the other hand, in IMET1, the lower growth rate, the reduced POE and the dramatically altered dependency of POE to pH change in the CA(g2209)-knockdown mutants under air-level CO2, which stays in sharp contrast to the apparent lack of prominent phenotypic response in the BCT (g1855)-knockdown mutants, underscored the crucial role of this CA gene in carbon assimilation and maintenance of pH homeostasis in this microalga. Together, these observations provide initial functional evidence for the array of genes that underlie carbon assimilation in Nannochloropsis spp. A combination of biochemical, cell biology and genetic (both forward and reverse) approaches would be required to further untangle the individual roles and interaction among these enzymes and transporters, which might hold the key for improving CO2-fixation efficiency and capability in microalgae. The efficiency, robustness and general applicability of the RNAi-based method, plus our simultaneously demonstrated Cas9-based genome editing method at the same strain (Wang etal., 2016a), raised the possibility of large-scale, individual-based gene function validation and screening for industrial microalgae. Such technical advances in metabolic engineering, plus those in non-invasive, label-free imaging, sorting and genotyping of microalgae at single-cell resolution (Ji etal., 2014; Wang etal., 2014b, 2016b; Zhang etal., 2015a,b; Teng etal., 2016), should greatly expedite the rational design and development of superior industrial strains for biofuel production and carbon sequestration. Nannochloropsis oceanica strain IMET1 was inoculated into modified f/2 liquid medium containing 30gL−1 sea salt (Realocean, USA), 10mm Tris-HCl (pH 7.6), 427.5mgL−1 NaNO3, 30mgL−1 NaH2PO4·H2O, 5mgL−1 trace metal mixture (4.36gL−1 Na2EDTA·2H2O, 3.15gL−1 FeCl3·6H2O, 10mgL−1 CoCl2·6H2O, 22mgL−1 ZnSO4·7H2O, 180mgL−1 MnCl2·4H2O, 9.8mgL−1 CuSO4·5H2O, 6.3mgL−1 Na2MoO4·2H2O) and 2.5mgL−1 vitamin stock solution (1mgL−1 vitamin B12, 1mgL−1 biotin, 200mgL−1 thiamine; Kang etal., 2015). The cells were cultivated in liquid cultures under continuous light (approximately 50±5μmol photonsm−2sec−1) at 25°C, and aerated by bubbling with either air or 5% CO2 (i.e. a mixture of air and CO2 at a volume ration of 95 to 5) at an aeration rate (i.e. gas flow) of 0.6 vvm (volume gas per volume medium per minute; 120mL gas 200mL cultures min−1). Nannochloropsis core genes of RNAi machinery were identified based on the previously published genome annotations. Those core genes of RNAi machinery from other algae were obtained by searching related proteins via BlastP and tBlastN against the proteome and the genome database from NCBI website (http://www.ncbi.nlm.nih.gov/protein/), respectively. Functional domains of RNAi pathway core components were detected using the ScanProsite tool (http://prosite.expasy.org/scanprosite/; de Castro etal., 2006) and Interproscan (http://www.ebi.ac.uk/interpro/search/sequence-search; Jones etal., 2014). For phylogenetic analysis, the amino acid sequences of proteins were edited in FASTA format and loaded into MEGA 4 software. Sequences were aligned using MUSCLE (Edgar, 2004), and phylogenetic trees were subsequently constructed in MEGA 4 using the neighbor-joining method with pairwise deletion (Tamura etal., 2007). Bootstrap value, the percentage of replicate trees in which the associated taxa clustered together, was tested for 1000 replicates. A 232-bp SF (corresponding to the CA nucleotide sequence from 38 to 269 bp) and a 447-bp LF (corresponding to the CA gene sequence from 38 to 484 bp) were amplified from the N.oceanica IMET1 cDNA, respectively, with the primers CA2209_fw (containing a EcoRI site) and CA2209_rv1 (containing a XbaI site), and CA2209_fw and CA2209_rv2 (containing a XbaI site; TableS2). These two fragments had the first 232 bp in common. The fragments were digested with EcoRI and XbaI, and ligated in sense and antisense orientations to the EcoRI site of the linearized phir-PtGUS vector to create phir-PtCA plasmid. The promoter region of β-tubulin of N.oceanica IMET1 was amplified from genomic DNA using the primers Notub_fw (containing a SacI site) and Notub_rv (containing a NcoI site; TableS2), then was digested with SacI and NcoI and ligated in the phir-PtCA plasmid replacing the P.tricornutum fcpB promoter to create phir-NoCA plasmid. Other RNAi expression vectors (phir-NoBCT and phir-NoCCMP1779-11263) for targeted BCT (g1855; SF: 221 bp, corresponding to the nucleotide sequence from 1282 to 1502 bp; LF: 434 bp, corresponding to the nucleotide sequence from 1282 to 1715 bp) and CCMP1779|11263 (11263; SF: 229 bp, corresponding to the nucleotide sequence from 1395 to 1623 bp; LF: 404 bp, corresponding to the CA nucleotide sequence from 1395 to 1798 bp) were constructed with corresponding primers (BCT1855_fw, BCT1855_rv1 and BCT1855_rv2; CCMP1779-11263_fw, CCMP1779-11263_rv1 and CCMP1779-11263_rv2; TableS2) containing EcoRI and XbaI sites (sense and antisense sequences) to replace the inverted repeat of CA in phir-NoCA plasmid. The IMET1 genes can be assessed at http://nanno.single-cell.cn/, whereas the CCMP1779 genes can be assessed at https://bmb.natsci.msu.edu/faculty/christoph-benning/nannochloropsis-oceanica-ccmp1779/. For electro-transformation, the expression cassettes were first amplified by PCR using primers (ECF and ECR; TableS2). The PCR products were purified using a MicroElute Cycle Pure kit (Omega Bio-tek, Norcross, GA, USA) and dissolved in ddH2O at 3–5μgμL−1 DNA concentration. All recovered DNA was stored at −20°C until subsequent use. Next, N.oceanica in the mid-exponential phase was harvested and concentrated to 108 cellsml−1. Then, microalgal cells were washed three-four times with pre-cooled 375mm sorbitol solution at 4°C and re-suspended in 375mm cold sorbitol solution. The cells were then electroporated in a 2-mm cuvette using an ECM630 BTX electroporator set at 500 Ω, 50μF and 2200V. Following electroporation, the cells were resuspended in 10mL modified f/2 media and incubated for 24h at 25±1°C with agitation at 50 rpm and 25μmol photons m−2s−1 before plating on f/2 plates containing 5μgmL−1 zeocin. After allowing the cells to recover under weak light, 10 mL medium containing the microalgal cells was centrifuged at 5000 g for 5min, and the cells were plated onto the agar plate with 5μgml−1 zeocin. Resistant colonies were observed after 2–3weeks, and picked after 4–5weeks. Genomic DNA extraction from WT and transformants of N.oceanica was performed using the E.Z.N.A.™ Plant DNA Kit (Omega Bio-tek). The incorporation of constructed gene expression cassettes into Nannochloropsis genomes was demonstrated by genomic DNA PCR. PCR was performed with primers (ble-F and ble-R; TableS2) of the ble gene. The PCR products were detected by agarose gel electrophoresis. The relative abundance of CA transcripts was determined by real-time qPCR. Total RNA was extracted from the microalgae using an E.Z.N.A.™ Plant RNA Kit (Omega Bio-Tek). The yield and purity of RNA were assessed by determination of the absorbance (Abs) at 260 and 280nm. The RNA was only used when the Abs260nm/Abs280nm ratio was 1.8. RNA integrity was assessed using a 1% agarose gel with the RNA 6000 Nano Assay Kit and Agilent 2100 Bioanalyzer. The extracted total RNA was stored at −70°C until further use. Elimination of DNA contamination was most successful during cDNA synthesis with the PrimeScript RT Reagent Kit (Takara-Bio, Tokyo, Japan) with gDNA Eraser. First strand cDNA was synthesized by reverse-transcribing 2μg total RNA with the PrimeScript®RT Reagent Kit. All cDNAs were stored at −20°C until use. RT-qPCR reactions were performed in 96-well optical reaction plates with a Roche LightCycle480 Real-Time PCR system using the manufacturer\'s kit (Roche, Rotkreuz, Switzerland) with 20μL mixture per well according to the manufacturer\'s directions. After 40 cycles, a melting curve analysis was conducted (60–95°C) to verify the specificity of the amplicons. Each amplification cycle was repeated three times. The specificity of the amplicons was confirmed by the presence of a single peak. The primers employed for CA(g2209), BCT(g1855) and CCMP1779|11263 were CA2209F and CA2209R, BCT1855F and BCT1855R, CCMP1779-11263F and CCMP1779-11263R, respectively, which are unique to the CA and BCT sequences (TableS2). The β-actin gene was used as a housekeeping marker control, and the primers used were ACT1F and ACT1R (TableS2). The Ct (threshold cycle) value for each well was measured and calculated. CA or BCT mRNA accumulation in transgenic and WT cells was quantified after normalization to β-actin gene transcripts. Relative expression level was calculated using the 2−ΔΔt method, where ΔΔt represents the value of [(Cttarget−Ctactin)mutant−(Cttarget−Ctactin)wild-type]. To test the stability of target gene silencing, the mutant and WT microalgal cells were cultured for 20days under air-level CO2 and then inoculated into fresh modified f/2 medium using a volume ratio of 1:10. The cycle was repeated for 18 rounds, which together span over 1year. Then the transcript level of targeted genes was interrogated using real-time qPCR. To probe the cytosine methylation of the targeted genes, the McrBC-PCR (MCR) method was used because the McrBC enzyme specifically cleaves methylated DNA (Fouse etal., 2010). Genomic DNA from the CA(g2209)-knockdown N.oceanica lines of M4 and M5 was digested overnight at 37°C with the McrBC enzyme (NEB, Hitchin, UK). The presence of methylated sites was probed by performing PCR on the coding region (the MCR2 and MCR3 fragments; Figure7a) and its upstream ~1.1kb region (the MCR1 fragment; Figure7a) and downstream ~0.6kb regions (the MCR4 fragment; Figure7a; primers listed in TableS3). As a negative control, the same amplification reaction was performed on DNA digested with McrBC without GTP as cofactor, as the McrBC enzyme requires GTP as cofactor. To further map the cytosine methylation pattern of the targeted gene, genomic DNA from the M4 mutant line was treated with bisulfite using the EpiMark® Bisulfite Conversion Kit (NEB). The sample was subsequently amplified by PCR with EpiMark® Hotstart Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA) using a number of different primer pairs that target various loci on both the sense and antisense strands (Figure7b; TableS3). The PCR products were, respectively, cloned to pMD19-T vector (Takara), and 10 clones from each PCR product were sequenced. Then the methylated positions were analyzed using the CyMATE software (Hetzl etal., 2007). To perform the spot test, an equal number of cells (2×104 cells) was cultured for 14days on agar plate containing modified f/2 medium under air-level CO2. For parameters related to photosynthesis, Chla content was measured using a modified extraction method (Ritchie, 2006). Briefly, a sample of the algal culture was centrifuged at 2500 g, and the filter was extracted in the dark for 24h in 100% ethanol saturated with MgCO3. The samples were then centrifuged and the supernatant analyzed spectrophotometrically. Extinction values at 632, 649, 665 and 750nm were recorded using a UV-Vis spectrophotometer. Chlorophyll fluorescence was measured using a pulse-amplitude modulated fluorometer (Image PAM, Walz, Effeltrich, Germany) to determine the quantum yield of photosystem II [Y(II)] when microalgal cells were cultured to the exponential phase. The oxygen evolution rate was determined with a Clark-type2 oxygen electrode (Hansatech, Norfolk, UK) and normalized to the Chla content of the N.oceanica culture. Microalgal cells of the CA(g2209)-knockdown and WT strains cultured in modified f/2 liquid medium under air-level CO2 were sampled at the log phase and centrifuged at 5000 rpm for 5min. After centrifugation, the cells were resuspended in fresh f/2 medium prepared with the buffers of various pH (either the pH 6.0 MES buffer, the pH 7.8 HEPES buffer, or the pH 9.0 APOS buffer) or without any buffer (pH 8.2, i.e. the pH of f/2 medium) for subsequent measurement of the photosynthetic O2 evolution rate. For quantification of the g2209 transcript via real-time qPCR, the cells were maintained in the buffers for 1 h, before proceeding to algal cell collection and RNA extraction. The complete annotated sequences of the expression cassette used for generating the RNAi-mediated knockdown mutants of N.oceanica IMET1 have been deposited under GenBank accession numbers of KX396597, KX426568 and KX787913. They are also accessible from the journal website (DataS1–S3). The authors are grateful to the anonymous reviewers for their valuable improvement to this manuscript. The authors thank Prof. Ansgar Poetsch for proofreading the English. The authors acknowledge support from National Natural Science Foundation of China (31425002 and 41576144), Ministry of Science and Technology of China (2012CB721101 and 2012AA02A707), Biological Carbon Sequestration Program (KSZD-EW-Z-017 and ZDRW-ZS-2016-3) from Chinese Academy of Sciences, and Young Investigator Program (ZR2015CQ003) from Natural Science Foundation of Shandong Province. JX and LW designed research; LW, YX and QW generated, screened and phenotyped knockdown mutants in N.oceanica IMET1 and CCMP1779; HH and JY constructed RNAi plasmids, and generated and screened mutants in CCMP1779; QW and YX assisted with transformation; LW performed DNA methylation site mapping experiments; the RNAi method in Nannochloropsis spp. originated from the lab of HH; LW, HH and JX analyzed the data and wrote the paper. tpj13411-sup-0001-FigS1.pptapplication/mspowerpoint, 227 KB FigureS1. Phylogenetic analysis of Nannochloropsis CAs. tpj13411-sup-0002-FigS2.pptapplication/mspowerpoint, 120 KB FigureS2. Temporal pattern of transcript abundance of the targeted genes in Nannochloropsis oceanica IMET1 under N+ and N− conditions. tpj13411-sup-0003-FigS3.pptapplication/mspowerpoint, 121.5 KB FigureS3. Transcript abundance of the other CAs in the CA(g2209)-knockdown lines of Nannochloropsis oceanica IMET1 (M4 and M5). tpj13411-sup-0004-FigS4.pptapplication/mspowerpoint, 117.5 KB FigureS4. Transcript abundance of CA(g2209) in the CA(g2209)-knockdown lines of Nannochloropsis oceanica IMET1 (M4 and M5) measured under different pH values. tpj13411-sup-0005-FigS5.pptapplication/mspowerpoint, 117.5 KB FigureS5. Temporal stability of gene silencing in the CA(g2209)-knockdown lines (M4 and M5) and BCT(g1855)-knockdown lines (BR3 and BR4) of Nannochloropsis oceanica IMET1. tpj13411-sup-0006-TableS1.docxWord document, 16.1 KB TableS1 CA homologs between Nannochloropsis oceanica strain IMET1 and strain CCMP1779 tpj13411-sup-0007-TableS2.docWord document, 40 KB TableS2 The primer sequences used for vector construction and qPCR in this study tpj13411-sup-0008-TableS3.docWord document, 36 KB TableS3 The nucleotide sequences of the primers used for McrBC-PCR and bisulfite sequencing tpj13411-sup-0009-DatasetS1.docxWord document, 16.6 KB DataS1. Complete annotated sequence of the expression cassette used for generating the RNAi-mediated CA(g2209)-knockdown mutants of Nannochloropsis oceanica IMET1 tpj13411-sup-0010-DatasetS2.docxWord document, 16.7 KB DataS2. Complete annotated sequence of the expression cassette used for generating the RNAi-mediated BCT(g1855)-knockdown mutants of Nannochloropsis oceanica IMET1. tpj13411-sup-0011-DatasetS3.docxWord document, 16.6 KB DataS3. Complete annotated sequence of the expression cassette used for generating the RNAi-mediated CA(11263)-knockdown mutants of Nannochloropsis oceanica CCMP1779. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Abida, H., Dolch, L.J., Mei, C. etal. (2015) Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum. Plant Physiol. 167, 118– 136. Armbrust, E.V., Berges, J.A., Bowler, C. etal. (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science, 306, 79– 86. Baker, N.R. 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