ABSTRACT
Synthetic biology is an exciting new area of research that combines science and engineering to design and build new biological functions and systems. Predictably, with the development of synthetic biology, more efficient and economical photosynthetic microalgae chassis will be successfully constructed, making it possible to break through laboratory research into large-scale industrial applications. The synthesis of a range of biochemicals has been demonstrated in cyanobacteria; however, low product titers are the biggest barrier to the commercialization of cyanobacterial biotechnology. This review summarizes the applied improvement strategies from the perspectives of cyanobacteria chassis cells and synthetic biology. The harvest advantages of cyanobacterial products and the latest progress in improving production strategies are discussed according to the product status. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in the application and development of cyanobacteria genetic tool kits in biochemical synthesis, environmental monitoring, and remediation were assessed.
ABSTRACT
It is a vital step to recognize cyanobacteria promoters on a genome-wide scale. Computational methods are promising to assist in difficult biological identification. When building recognition models, these methods rely on non-promoter generation to cope with the lack of real non-promoters. Nevertheless, the factitious significant difference between promoters and non-promoters causes over-optimistic prediction. Moreover, designed for E. coli or B. subtilis, existing methods cannot uncover novel, distinct motifs among cyanobacterial promoters. To address these issues, this work first proposes a novel non-promoter generation strategy called phantom sampling, which can eliminate the factitious difference between promoters and generated non-promoters. Furthermore, it elaborates a novel promoter prediction model based on the Siamese network (SiamProm), which can amplify the hidden difference between promoters and non-promoters through a joint characterization of global associations, upstream and downstream contexts, and neighboring associations w.r.t. k-mer tokens. The comparison with state-of-the-art methods demonstrates the superiority of our phantom sampling and SiamProm. Both comprehensive ablation studies and feature space illustrations also validate the effectiveness of the Siamese network and its components. More importantly, SiamProm, upon our phantom sampling, finds a novel cyanobacterial promoter motif ('GCGATCGC'), which is palindrome-patterned, content-conserved, but position-shifted.
Subject(s)
Cyanobacteria , Promoter Regions, Genetic , Cyanobacteria/genetics , Computational Biology/methods , AlgorithmsABSTRACT
OBJECTIVE: This systematic review and meta-analysis investigated whether the use of azithromycin during labour or caesarean section reduces the incidence of sepsis and infection among mothers and newborns. DATA SOURCES: We independently searched the PubMed, Web of Science, Cochrane Library and EMBASE databases for relevant studies published before February, 2024. METHODS: We included RCTs that evaluated the effect of prenatal oral or intravenous azithromycin or placebo on intrapartum or postpartum infection incidence. We included studies evaluating women who had vaginal births as well as caesarean sections. Studies reporting maternal and neonatal infections were included in the current analysis. Review Manager 5.4 was used to analyse 6 randomized clinical trials involving 44,448 mothers and 44,820 newborns. The risk of bias of each included study was assessed using the criteria outlined in the Cochrane Handbook for Systematic Reviews of Interventions.Primary outcomes included the incidence of maternal sepsis and all-cause mortality and neonatal sepsis and all-cause mortality; secondary outcomes included maternal (endometritis, wound and surgical site infections, chorioamnionitis, and urinary tract infections) and neonatal outcomes (infections of the eyes, ears and skin). A random-effects model was used to test for overall effects and heterogeneity. RESULTS: The pooled odds ratios (ORs) were as follows: 0.65 for maternal sepsis (95% CI, 0.55-0.77; I2, 0%; P < .00001); 0.62 for endometritis (95% CI, 0.52-0.74; I2, 2%; P < .00001); and 0.43 for maternal wound or surgical site infection (95% CI, 0.24-0.78; P < .005); however, there was great heterogeneity among the studies (I2, 75%). The pooled OR for pyelonephritis and urinary tract infections was 0.3 (95% CI, 0.17-0.52; I2, 0%; P < .0001), and that for neonatal skin infections was 0.48 (95% CI, 0.35-0.65; I2, 0%, P < .00001). There was no significant difference in maternal all-cause mortality or incidence of chorioamnionitis between the two groups. No significant differences were observed in the incidence of neonatal sepsis or suspected sepsis, all-cause mortality, or infections of the eyes or ears. CONCLUSION: In this meta-analysis, azithromycin use during labour reduced the incidence of maternal sepsis, endometritis, incisional infections and urinary tract infections but did not reduce the incidence of neonatal-associated infections, except for neonatal skin infections. These findings indicate that azithromycin may be potentially beneficial for maternal postpartum infections, but its effect on neonatal prognosis remains unclear. Azithromycin should be used antenatally only if the clinical indication is clear and the potential benefits outweigh the harms.
Subject(s)
Anti-Bacterial Agents , Azithromycin , Randomized Controlled Trials as Topic , Humans , Azithromycin/therapeutic use , Azithromycin/administration & dosage , Female , Pregnancy , Infant, Newborn , Anti-Bacterial Agents/therapeutic use , Incidence , Labor, Obstetric , Cesarean Section/statistics & numerical data , Puerperal Infection/prevention & control , Puerperal Infection/epidemiology , Endometritis/prevention & control , Endometritis/epidemiology , Sepsis/prevention & control , Sepsis/epidemiology , Neonatal Sepsis/prevention & control , Neonatal Sepsis/epidemiologyABSTRACT
Polybrominated diphenyl ether (PBDE) contamination is common in aquatic environments and can severely damage aquatic organisms. However, there is a lack of information on the response and self-adaptation mechanisms of these organisms. Chlorella pyrenoidosa was treated with 2,2',4,4'-tetrabromodiphenyl ether (BDE47), causing significant growth inhibition, pigment reduction, oxidative stress, and chloroplast atrophy. Photosynthetic damage contributed to inhibition, as indicated by Fv/Fm, Chl a fluorescence induction, photosynthetic oxygen evolution activity, and photosystem subunit stoichiometry. Here, Chl a fluorescence induction and quinone electron acceptor (QA-) reoxidation kinetics showed that the PSII donor and acceptor sides were insensitive to BDE47. Quantitative analyses of D1 and PsaD proteins illustrated that PSII and PSI complexes were the main primary targets of photosynthesis inhibition by BDE47. Significant modulation of PSII complex might have been caused by the potential binding of BDE47 on D1 protein, and molecular docking was performed to investigate this. Increased activation of antioxidant defense systems and photosystem repair as a function of exposure time indicated a positive resistance to BDE47. After a 5-day exposure, 23 % of BDE47 was metabolized. Our findings suggest that C. pyrenoidosa has potential as a bioremediator for wastewater-borne PBDEs and can improve our understanding of ecological risks to microalgae.
Subject(s)
Chlorella , Halogenated Diphenyl Ethers , Halogenated Diphenyl Ethers/toxicity , Halogenated Diphenyl Ethers/metabolism , Chlorella/metabolism , Molecular Docking Simulation , Photosynthesis , Electron Transport , Photosystem II Protein Complex/metabolismABSTRACT
Even though sunlight energy far outweighs the energy required by human activities, its utilization is a key goal in the field of renewable energies. Microalgae have emerged as a promising new and sustainable feedstock for meeting rising food and feed demand. Because traditional methods of microalgal improvement are likely to have reached their limits, genetic engineering is expected to allow for further increases in the photosynthesis and productivity of microalgae. Understanding the mechanisms that control photosynthesis will enable researchers to identify targets for genetic engineering and, in the end, increase biomass yield, offsetting the costs of cultivation systems and downstream biomass processing. This review describes the molecular events that happen during photosynthesis and microalgal productivity through genetic engineering and discusses future strategies and the limitations of genetic engineering in microalgal productivity. We highlight the major achievements in manipulating the fundamental mechanisms of microalgal photosynthesis and biomass production, as well as promising approaches for making significant contributions to upcoming microalgal-based biotechnology.
Subject(s)
Microalgae , Humans , Microalgae/genetics , Genetic Engineering , Biotechnology/methods , Photosynthesis/genetics , Renewable Energy , Biomass , BiofuelsABSTRACT
The persistent organic pollutant perfluorooctanoic acid (PFOA) is ubiquitous in aquatic environments. However, little is known about its toxicity to microalgae or the mechanisms by which they may self-adapt to it. We found that growth of the bloom-forming cyanobacterium Microcystis aeruginosa was initially inhibited, with inhibition attenuated after 12 d of PFOA exposure. Growth inhibition gradually decreased and stabilized over time. With increasing PFOA concentration, reactive oxygen species levels and superoxide dismutase and photosystem II activity significantly increased, while respiration, NDH-1 activity, and total carbohydrate content significantly decreased. Self-adaptation mechanisms included antioxidant pathways, energy transfer and distribution of photosystems, and repair of the PSI and NDH complexes. The patterns of change in these parameters were consistent with those of the expression levels of genes in their associated metabolic pathways. Our data suggest that PSII overcompensation might be a strategy by which M. aeruginosa contends with oxidative stress induced by PFOA. Multiple downstream photosynthesis-related proteins were upregulated as a function of PFOA exposure time. These findings may help elucidate physiological, genetic stress and self-adaptive responses of microalgae to PFOA exposure.
Subject(s)
Cyanobacteria , Microcystis , Photosynthesis , Oxidative Stress , Cyanobacteria/metabolismABSTRACT
The control of harmful cyanobacterial blooms has been becoming a global challenge. The development of eco-friendly algicides with strong specificity is urgently needed. The photosynthetic apparatus is a promising target site for algicides to minimize the possible harmful effects on animals and humans. In this study, biologically derived 2-hydroxychalcone efficiently inhibited the growth of bloom-forming M. aeruginosa by selectively interfering with photosynthesis. 2-Hydroxychalcone targeting Photosystem II (PSII) inhibited electron transfer between the primary and secondary electron acceptors (QA and QB) and the binding of plastoquinone (PQ) molecules to the QB binding pocket at the acceptor side of PSII, as revealed by polyphasic chlorophyll (Chl) a fluorescence induction and QA- reoxidation kinetics. Molecular docking for 2-hydroxychalcone to D1 protein and the proteomic responses of M. aeruginosa suggested that 2-hydroxychalcone formed a stable monodentate ligand with the nonheme iron in D1 protein, provoking significant modulation of PSII proteins. The unique binding mode of 2-hydroxychalcone with PSII differentiated it from classical PSII inhibitors. Furthermore, 2-hydroxychalcone down-regulated the expression of microcystin (MC) synthesis-related genes to restrain MC synthesis and release. These results indicated the potential application of 2-hydroxychalcone as an algicide or a template scaffold for designing novel derivatives with superior algicidal activity.
Subject(s)
Proteomics , Humans , Molecular Docking SimulationABSTRACT
The potential accumulation of chlorinated organophosphorus flame retardants (Cl-OPFRs) in aquatic environments sparked interest in studying the effects of Cl-OPFRs on cyanobacterial blooms. In this work, two common Cl-OPFRs, tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and tris(2-chloroethyl) phosphate (TCEP), induced dose-dependent biphasic effect on bloom-forming M. aeruginosa. The hormetic response to low-dose Cl-OPFRs was associated with the upregulation of the type I NADH dehydrogenase (NDH-1) complex and its mediated cyclic electron transfer (CET) pathway, as reflected by a transient post-illumination increase in chlorophyll fluorescence, the dark reduction of P700+ and the change of NDH-1-related gene expression. The increased CET activity and carotenoid content jointly reduced the intracellular ROS production, facilitating cyanobacterial growth. Conversely, a higher concentration of both Cl-OPFRs induced severe inhibition of growth and photosynthetic oxygen-evolving activity through an imbalance between PSII and PSI. Toxic-dose Cl-OPFRs inhibited state transition and fixed cells into the State I with a higher PSII/PSI ratio, as indicated by chlorophyll fluorescence induction, 77 K fluorescence emission spectra and photosystem stoichiometry. The elevated PSII/PSI ratio created an imbalance between the two photosystems and eventually lead to ROS overproduction, which generate adverse effects on cell growth. This work provides important insights into the hormetic mechanism of Cl-OPFRs on Microcystis aeruginosa and their potential roles in harmful cyanobacteria blooms.
Subject(s)
Flame Retardants , Microcystis , Chlorophyll , Flame Retardants/toxicity , Organophosphates , Organophosphorus Compounds/toxicity , Phosphates , Reactive Oxygen SpeciesABSTRACT
Oral supplemented nutraceuticals derived from food sources are surmised to improve the human health through interaction with the gastrointestinal bacteria. However, the lack of fundamental quality control and authoritative consensus (e.g., formulation, route of administration, dose, and dosage regimen) of these non-medical yet bioactive compounds are one of the main practical issues resulting in inconsistent individual responsiveness and confounded clinical outcomes of consuming nutraceuticals. Herein, we studied the dose effects of widely used food supplement, microalgae spirulina (Arthrospira platensis), on the colonic microbiota and physiological responses in healthy male Balb/c mice. Based on the analysis of 16s rDNA sequencing, compared to the saline-treated group, oral administration of spirulina once daily for 24 consecutive days altered the diversity, structure, and composition of colonic microbial community at the genus level. More importantly, the abundance of microbial taxa was markedly differentiated at the low (1.5 g/kg) and high (3.0 g/kg) dose of spirulina, among which the relative abundance of Clostridium XIVa, Desulfovibrio, Eubacterium, Barnesiella, Bacteroides, and Flavonifractor were modulated at various degrees. Evaluation of serum biomarkers in mice at the end of spirulina intervention showed reduced the oxidative stress and the blood lipid levels and increased the level of appetite controlling hormone leptin in a dose-response manner, which exhibited the significant correlation with differentially abundant microbiota taxa in the cecum. These findings provide direct evidences of dose-related modulation of gut microbiota and physiological states by spirulina, engendering its future mechanistic investigation of spirulina as potential sources of prebiotics for beneficial health effects via the interaction with gut microbiota.
Subject(s)
Cecum/drug effects , Colon/drug effects , Dietary Supplements/analysis , Gastrointestinal Microbiome/drug effects , Spirulina/chemistry , Animals , Bacteroides/classification , Bacteroides/genetics , Bacteroides/isolation & purification , Bacteroidetes/classification , Bacteroidetes/genetics , Bacteroidetes/isolation & purification , Cecum/microbiology , Clostridiales/classification , Clostridiales/genetics , Clostridiales/isolation & purification , Clostridium/classification , Clostridium/genetics , Clostridium/isolation & purification , Colon/microbiology , Complex Mixtures/administration & dosage , Desulfovibrio/classification , Desulfovibrio/genetics , Desulfovibrio/isolation & purification , Dose-Response Relationship, Drug , Eubacterium/classification , Eubacterium/genetics , Eubacterium/isolation & purification , Feces/microbiology , Gastrointestinal Microbiome/genetics , Leptin/blood , Lipids/blood , Male , Mice , Mice, Inbred BALB C , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNAABSTRACT
UV radiation is a serious threat to life, and algae have developed highly efficient adaptations to UV radiation through the course of evolution. To date, studies investigating the mechanisms of UV adaptation in algae have focused on physiological regulation and associated protein coding genes, with only a few reports on associated protein non-coding genes. In a previous study, we found that Cre-miR914 was significantly down-regulated in Chlamydomonas reinhardtii in response to heat shock. In the present study, we aimed to determine whether Cre-miR914 plays a role in response to UV-B radiation. Our bioinformatics analysis indicated that the potential target gene of Cre-miR914 is ribosomal protein L18 (RPL18). We also measured the expression of Cre-miR914 and RPL18 in response to UV-B radiation through qPCR analysis. Then, we constructed cell lines overexpressing Cre-miR914 or RPL18, and performed survival experiments under UV-B stress. The results showed that Cre-miR914 overexpression decreased resistance while RPL18 overexpression enhanced tolerance to UV-B radiation. These results indicate that Cre-miR914 and its potential target gene RPL18 are involved in the adaptation to UV-B in C. reinhardtii.
Subject(s)
Chlamydomonas reinhardtii/radiation effects , MicroRNAs/physiology , Plant Proteins/metabolism , Ribosomal Proteins/metabolism , Cell Line , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/metabolism , Chlamydomonas reinhardtii/physiology , Genes, Plant/genetics , Genes, Plant/physiology , MicroRNAs/genetics , MicroRNAs/metabolism , Photosynthesis , Plant Proteins/physiology , Radiation Tolerance/genetics , Radiation Tolerance/physiology , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain Reaction , Ribosomal Proteins/physiology , Ultraviolet RaysABSTRACT
As the transcriptional and post-transcriptional regulators of gene expression, small RNAs (sRNAs) play important roles in every domain of life in organisms. It has been discovered gradually that bacteria possess multiple means of gene regulation using RNAs. They have been continuously used as model organisms for photosynthesis, metabolism, biotechnology, evolution, and nitrogen fixation for many decades. Cyanobacteria, one of the most ancient life forms, constitute all kinds of photoautotrophic bacteria and exist in almost any environment on this planet. It is believed that a complex RNA-based regulatory mechanism functions in cyanobacteria to help them adapt to changes and stresses in diverse environments. Although lagging far behind other model microorganisms, such as yeast and Escherichia coli, more and more non-coding regulatory sRNAs have been recognized in cyanobacteria during the past decades. In this article, by focusing on cyanobacterial sRNAs, the approaches for detection and targeting of sRNAs will be summarized, four major mechanisms and regulatory functions will be generalized, eight types of cis-encoded sRNA and four types of trans-encoded sRNAs will be reviewed in detail, and their possible physiological functions will be further discussed.
ABSTRACT
Pili are found on the surface of many bacteria and play important roles in cell motility, pathogenesis, biofilm formation, and sensing and reacting to environmental changes. Cell motility in the model cyanobacterium Synechocystis sp. PCC 6803 relies on expression of the putative pilA9-pilA10-pilA11-slr2018 operon. In this study, we identified the antisense RNA PilR encoded in the noncoding strand of the prepilin-encoding gene pilA11. Analysis of overexpressor [PilR(+)] and suppressor [PilR(-)] mutant strains revealed that PilR is a direct negative regulator of PilA11 protein. Although overexpression of PilR did not affect cell growth, it greatly reduced levels of pilA11 mRNA and protein and decreased both the thickness and number of pili, resulting in limited cell motility and small, distinct colonies. Suppression of PilR had the opposite effect. A hypothetical model on the regulation of pilA9-pilA10-pilA11-slr2018 operon expression by PilR was proposed. These results add a layer of complexity to the mechanisms controlling pilA11 gene expression and cell motility, and provide novel insights into how sRNA and the intergenic region secondary structures can work together to discoordinatly regulate target gene in an operon in cyanobacterium.
ABSTRACT
Small regulatory RNAs (sRNAs) function as transcriptional and post-transcriptional regulators of gene expression in organisms from all domains of life. Cyanobacteria are thought to have developed a complex RNA-based regulatory mechanism. In the current study, by genome-wide analysis of differentially expressed small RNAs in Synechocystis sp. PCC 6803 under high light conditions, we discovered an asRNA (RblR) that is 113nt in length and completely complementary to its target gene rbcL, which encodes the large chain of RuBisCO, the enzyme that catalyzes carbon fixation. Further analysis of the RblR(+)/(-) mutants revealed that RblR acts as a positive regulator of rbcL under various stress conditions; Suppressing RblR adversely affects carbon assimilation and thus the yield, and those phenotypes of both the wild type and the overexpressor could be downgraded to the suppressor level by carbonate depletion, indicated a regulatory role of RblR in CO2 assimilation. In addition, a real-time expression platform in Escherichia coli was setup and which confirmed that RblR promoted the translation of the rbcL mRNA into the RbcL protein. The present study is the first report of a regulatory RNA that targets RbcL in Synechocystis sp. PCC 6803, and provides strong evidence that RblR regulates photosynthesis by positively modulating rbcL expression in Synechocystis.
ABSTRACT
MicroRNAs play an important role in abiotic stress responses in higher plants and animals, but their role in stress adaptation in algae remains unknown. In this study, the expression of identified and putative miRNAs in Chlamydomonas reinhardtii was assessed using quantitative polymerase chain reaction; some of the miRNAs (Cre-miR906-3p) were up-regulated, whereas others (Cre-miR910) were down-regulated when the species was subjected to multiple abiotic stresses. With degradome sequencing data, we also identified ATP4 (the d-subunit of ATP synthase) and NCR2 (NADPH: cytochrome P450 reductase) as one of the several targets of Cre-miR906-3p and Cre-miR910, respectively. Q-PCR data indicated that ATP4, which was expressed inversely in relation to Cre-miR906-3p under stress conditions. Overexpressing of Cre-miR906-3p enhanced resistance to multiple stresses; conversely, overexpressing of ATP4 produced the opposite effect. These data of Q-PCR, degradome sequencing and adaptation of overexpressing lines indicated that Cre-miR906-3p and its target ATP4 were a part of the same pathway for stress adaptation. We found that Cre-miR910 and its target NCR2 were also a part of this pathway. Overexpressing of Cre-miR910 decreased, whereas that of NCR2 increased the adaption to multiple stresses. Our findings suggest that the two classes of miRNAs synergistically mediate stress adaptation in algae.
Subject(s)
Chlamydomonas reinhardtii/metabolism , Gene Expression Regulation, Plant/physiology , MicroRNAs/biosynthesis , RNA, Plant/biosynthesis , Stress, Physiological/physiology , Chlamydomonas reinhardtii/genetics , MicroRNAs/genetics , Plant Proteins/biosynthesis , Plant Proteins/genetics , RNA, Plant/geneticsABSTRACT
We previously showed that both the linear photosynthetic electron transportation rate and the respiration rate dropped significantly during N starvation-induced neutral lipid accumulation in an oil-producing microalga, Chlorella sorokiniana, and proposed a possible role for cyclic electron flow (CEF) in ATP supply. In this study, we further exploited this hypothesis in both Chlorella sorokiniana C3 and the model green alga Chlamydomonas. We found that both the rate of CEF around photosystem I and the activity of thylakoid membrane-located ATP synthetase increased significantly during N starvation to drive ATP production. Furthermore, we demonstrated that the Chlamydomonas mutant pgrl1, which is deficient in PGRL1-mediated CEF, accumulated less neutral lipids and had reduced rates of CEF under N starvation. Further analysis revealed that Ca(2+) signaling regulates N starvation-induced neutral lipid biosynthesis in Chlamydomonas by increasing calmodulin activity and boosting the expression of the calcium sensor protein that regulates Pgrl1-mediated CEF. Thus, Ca(2+)-regulated CEF supplies ATP for N starvation-induced lipid biosynthesis in green alga. The increased CEF may re-equilibrate the ATP/NADPH balance and recycle excess light energy in photosystems to prevent photooxidative damage, suggesting Ca(2+)-regulated CEF also played a key role in protecting and sustaining photosystems.
Subject(s)
Adenosine Triphosphate/metabolism , Calcium/metabolism , Chlorophyta/metabolism , Lipids/biosynthesis , Nitrogen/metabolism , ATP Synthetase Complexes/metabolism , Algal Proteins/genetics , Algal Proteins/metabolism , Catalase/genetics , Catalase/metabolism , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/metabolism , Chlorella/classification , Chlorella/genetics , Chlorella/metabolism , Chlorophyta/genetics , Electron Transport/genetics , Electron Transport/physiology , Immunoblotting , Microscopy, Confocal , Mutation , Photosynthesis/genetics , Photosynthesis/physiology , Photosystem I Protein Complex/metabolism , Phylogeny , Reverse Transcriptase Polymerase Chain Reaction , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Thylakoids/enzymology , Thylakoids/metabolismABSTRACT
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 plastids 1) and the flagellar protein CDPK3 (calcium-dependent protein kinase 3). Adding an intron from RBCS2 (ribulose bisphosphate carboxylase/oxygenase small subunit 2) 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.