Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-st...Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.展开更多
Species in genus Nannochloropsis,especially N.oceanica and N.gaditana,have been evolving as the model microalgae for both application and theory studies.The position effect of genome integration,the carrying capabilit...Species in genus Nannochloropsis,especially N.oceanica and N.gaditana,have been evolving as the model microalgae for both application and theory studies.The position effect of genome integration,the carrying capability limitation of integrative vectors and the instability of non-integrative vectors have hindered Nannochloropsis genetic modification with concatenate genes and extremely long DNA fragments.The molecular tools including genetic transformation,homologous recombination,gene edition,gene stacking and episome vectors for transient gene expression and diverse reporters and selection markers have been rapidly developing in Nannochloropsis species.The construction of animal and plant artificial chromosomes with“top down”strategy has set fine examples for the construction of Nannochloropsis artificial chromosomes(NannoACs).It seems that the methods and materials to set the foundation for constructing NannoACs are at hands.In this review,we outlined the current status of transgenes in Nannochloropsis species,summarized the limitations of both integrative and non-integrative vectors,and proposed a tentative approach to construct NannoACs by doubling and stabilizing the genome first,and then truncating the natural chromosomes.NannoACs once constructed will facilitate transferring the desired traits and concatenate genes into Nannochloropsis genetic backgrounds,thus contributing towards its genetic improvement and synthetic biological studies.展开更多
Zeocin can cause double strand breaks of DNA and thus is frequently used as a selective antibiotic of eukaryotic Sh ble transformants. In non-transformation system, Zeocin may function as a mutagen if not totally leth...Zeocin can cause double strand breaks of DNA and thus is frequently used as a selective antibiotic of eukaryotic Sh ble transformants. In non-transformation system, Zeocin may function as a mutagen if not totally lethal. To verify such function of Zeocin, we mutated Nannochloropsis oceanica by increasing the concentration of Zeocin in medium gradually, and isolated a N. oceanica strain(single cell culture) which survived Zeocin up to 10.0μg mL^(-1). The Zeocin-tolerant strain entered the exponential growth phase later and grew slower than the wild strain. Transcriptome profiling showed that the Zeocin-tolerant N. oceanica strain survived Zeocin mainly by adapting(heritable), rather than acclimating(plastic) to Zeocin. Hence mutating N. oceanica with Zeocin was approved effective. Meanwhile, the physiological characteristics of this Zeocin-tolerant strain were demonstrated. As we proposed, N. oceanica tolerated Zeocin by strengthening its protein degradation and antioxidation. The genes controlling cell division and cellular response to stimuli may also have played important roles in the reduction of growth and the tolerance to Zeocin. Our findings evidenced that Zeocin can serve as an appropriate mutagen of microalgae. Creating variations through mutation with Zeocin may help to study the genetic basis of the traits of this monoploidy and asexual microalga, as well as improve its production.展开更多
The marine microalga Nannochloropsis sp.contains various elongases and desaturases that are critical for biosynthesis of polyunsaturated fatty acids.A full-length cDNA encoding a long-chain fatty acid elongase,named N...The marine microalga Nannochloropsis sp.contains various elongases and desaturases that are critical for biosynthesis of polyunsaturated fatty acids.A full-length cDNA encoding a long-chain fatty acid elongase,named Ns FAE,was cloned from Nannochloropsis sp..The open reading frame of Ns FAE(GenBank accession no.MF680548)consisted of 1068 bp and encoded a predicted protein of 355 amino acids with molecular mass 38.8 k Da.The deduced polypeptide showed 43%–44%identity to fatty acyl elongases from other algae.RT-PCR experiments indicated that the Ns FAE gene exhibited the highest expression in Nannochloropsis sp.at 72 h(i.e.,during the third growth stage)and the expression was significantly lower in the other four growth stages.Plasmid p Ns FAE-CRISPR and a recombinant DNA fragment(ADH1p-Ns FAE-CYCt)were transformed into Saccharomyces cerevisiae strain BY4742 using the CRISPR-Cas system.Yeast transformants containing Ns FAE produced three fatty acids not normally present in wild-type BY4742-linoleic acid,linolenic acid and eicosadienoic acid-indicating that Ns FAE encodes a functional elongase enzyme.展开更多
Marine microalga Nannochloropsis oceanica LAMB0001 were domesticated (~730 generations,~two days each) to adapt freshwater BG11 medium. A number of freshwater medium adapted colony-derived strains were obtained. The s...Marine microalga Nannochloropsis oceanica LAMB0001 were domesticated (~730 generations,~two days each) to adapt freshwater BG11 medium. A number of freshwater medium adapted colony-derived strains were obtained. The strains were verified phylogenetically to be N. oceanica LAMB0001 based on the 18S ribosomal RNA gene. Freshwater-medium adapted strain (FA1) grew faster in the BG11 medium prepared with freshwater than wild-type N. oceanica grew in f/2 medium prepared with seawater. We assumed that (1) the expression patterns of the genes that expressed differentially between FA1 and the wild-type N. oceanica exposing to the BG11 medium (WT-F) have been reprogrammed;(2) the physiological processes in which these genes involved have been modified;and (3) a Gene Ontology (GO) term or a KEGG pathway enriched by DEGs between FA1 and WT-F has been up- or down-regulated if it was enriched simultaneously by up- or down-regulated DEGs between FA1 and WT-F, respectively. Under these assumptions, we found that FA1 reprogrammed the expression patterns of a set of genes that involved in cell adhesion, membrane and membrane integrity, material transportation, cell movement, and cellular signaling network. These changes in cellular functions and metabolic pathways indicate that the microalga modified its gene expression pattern in a wide function range and at a high regulation rank in order to adapt to the freshwater medium. It is feasible to domesticate marine microalgae to a freshwater habitat, which may aid to modify their cultivation performances.展开更多
In this study, the RNA sequencing was used to describe the response of Nannochloropsis oceanica, a marine microalga, to benzo[a]pyrene(BaP), a polycyclic aromatic hydrocarbon, in order to elucidate the metabolic pathw...In this study, the RNA sequencing was used to describe the response of Nannochloropsis oceanica, a marine microalga, to benzo[a]pyrene(BaP), a polycyclic aromatic hydrocarbon, in order to elucidate the metabolic pathways(or processes) involved in microalgal response to this stubborn pollutant. N. oceanica was exposed to BaP at a concentration of 90 μg L^-1 for 72 h, and its transcriptome was sequenced through the Illumina HiSeq^TM 2500 platform. This concentration of BaP was selected as it is the lowest for modeling the most appropriate growth inhibition of N. oceanica for transcriptomic analysis. We found that N. oceanica responds to BaP through degrading proteins and repairing DNA damaged by BaP. In addition, superoxide dismutase(SOD) strengthened its performance by increasing its transcript abundance. The physiological mechanism underlining the response of N. oceanica to BaP as revealed by transcriptomic analysis was consistent with the biochemical insights documented previously.展开更多
In order to decipher the hygromycin B tolerance and resistance mechanisms of Nannochloropsis oceanica,the transcriptome profiles of a transgenic strain carrying a randomly integrated hygromycin B resistant gene,a hygr...In order to decipher the hygromycin B tolerance and resistance mechanisms of Nannochloropsis oceanica,the transcriptome profiles of a transgenic strain carrying a randomly integrated hygromycin B resistant gene,a hygromycin B-adaptive strain and a wild type strain of N.oceanica were compared by transcriptome sequencing(RNA-seq)without referring to a high quality genome sequence.The results showed that the adaptive strain adapts to the hygromycin B existing environments mainly by intensifying the expressions of the efflux pump ABC and MFS superfamily transporter genes,thus reducing the intracellular concentration of hygromycin B.The transgenic strain obtains the hygromycin B resistance ability solely by expressing exogenous resistance gene.Accordingly,the screening and maintenance of N.oceanica transformants should be carried out at an antibiotics concentration higher than the adaptive threshold.Our findings can help the genetic modification of N.oceanica with the application of hygromycin B.展开更多
Nannochloropsis is a genus of marine eukaryotic unicellular algae,which belongs to class Eustigmatophyceae.The spe-cies of Nannochloropsis which are fine rotifer feed and rich in eicosapentaenoic acid(EPA)are economic...Nannochloropsis is a genus of marine eukaryotic unicellular algae,which belongs to class Eustigmatophyceae.The spe-cies of Nannochloropsis which are fine rotifer feed and rich in eicosapentaenoic acid(EPA)are economically important.Species in this genus are usually 2-5μm in size and are morphologically similar,which makes their identification difficult.We obtained a monoclone of Nannochloropsis with plating method in this study.DNA was extracted and the quality was determined by restriction enzyme digestion and spectrophotometer analysis.The DNA extracted was used to amplify the sequences of 18S ribosomal RNA gene,ITS region of ribosomal RNA transcription unit and rbcL gene.The phylogenetic analysis was carried out by constructing the neighbor-joining trees with Tamura-Nei distances.The phylogenetic analysis showed that the monoclone is N.oceanica.展开更多
This study determined growth and lipid accumulation in Nannochloropsis sp. MASCC 11 cultivated at different pH, temperatures, and CO2 concentrations in 10-day period. The suitability for biodiesel production was also ...This study determined growth and lipid accumulation in Nannochloropsis sp. MASCC 11 cultivated at different pH, temperatures, and CO2 concentrations in 10-day period. The suitability for biodiesel production was also evaluated based on the fatty acid profiles of microalgae lipid. Nannochloropsis sp. MASCC 11 showed an excellent tolerance to acidic pH(as low as 4.0), high temperatures(at least 40℃), and high CO2 concentrations(5%-15%), which are major stressed conditions in flue gas. The highest algal biomass was acquired at pH of 9.0(0.44 g L^-1), a temperature of 35℃(0.63 g L^-1), and a CO2 concentration of 5%(2.27 g L^-1). Maximum lipid production was obtained at p H of 6.0(108.2 mg L^-1), a temperature of 35℃(134.6 mg L^-1), and a CO2 concentration of 5%(782.7 mg L^-1). Synthesis of polyunsaturated fatty acids(PUFAs) in biomass was stimulated under high CO_2 concentrations, remaining above 80% of total fatty acids, mainly composed of C16:3, C18:2, and C18:3. This led to the algae-based biodiesel having a lower oxidation stability, better cold flow properties, and other parameters such as its kinematic viscosity, cetane number, and specific gravity complied with ASTM or EN 14214 biodiesel specifications. Therefore, the improvement of oxidative stability needs to be considered before Nannochloropsis sp. MASCC 11 lipid can be used for biodiesel production, even if this species can grow well under stressful conditions.展开更多
Nannochloropsis oceanica is a marine microalgal species with both economic value and biological importance.It grows fast,contains rich oils,reproduces asexually,holds a small and haploidy genome,and is easy to be modi...Nannochloropsis oceanica is a marine microalgal species with both economic value and biological importance.It grows fast,contains rich oils,reproduces asexually,holds a small and haploidy genome,and is easy to be modified genetically.However,the genetic study of N.oceanica is scarce.Very less genetic bases of its traits have been deciphered,and no gene has been isolated from it with the function verified simultaneously via either genetic or reverse genetic approaches or both(de novo cloned).Changing medium salinity may aid to control harmful organisms met during large scale cultivation.As a stress,it may also facilitate the accumulation of desirable chemicals including fatty acids.In order to decipher the genetic basis of the low salinity tolerance of N.oceanica,we mutated N.oceanica with Zeocin.In total,five mutant bulks were constructed at equal number of cells,100 mutants each,which were tolerant to a discontinuous serial of salinities from that of 100%of f/2 to that of a mixture of 4%of f/2 and 94%of BG11.The bulks were genotyped through whole genome re-sequencing and analyzed with bulked mutant analysis(BMA)newly modified from bulked segregant analysis(BSA).In total,47 SNPs and 112 InDels were found to associate with the low salinity tolerance,and around them a set of low salinity tolerance associating genes were identified.A set of annotatable genes commonly found between control and different salinities indicated that the genes functioning in gene expression,energy metabolism and cellular structure may be involved in the low salinity tolerance.These associating molecular markers and genes around them were not enough for outlining the physiological mechanism underlining the tolerance;however they should aid to improve N.oceanica genetically.展开更多
The effects of the nitrogen sources sodium nitrate (NaNO3) and urea (CH4N2O) on growth, lipid production, and fatty acid composition of Nannochloropsis oceanica IMET1 were investigated. Nitrogen source affected cell d...The effects of the nitrogen sources sodium nitrate (NaNO3) and urea (CH4N2O) on growth, lipid production, and fatty acid composition of Nannochloropsis oceanica IMET1 were investigated. Nitrogen source affected cell density, dry cell weight, and lipid production. Cells grown in the nitrate medium increased dry cell weight and lipid weight in comparison with cells grown in the urea medium. The composition of fatty acids varied with nitrogen sources. IMET1cultured in the nitrate medium mainly contained C18:2 (14.9%) and C16:0 (6.3%) fatty acids, while IMET1 in the urea medium mainly contained C22:0 (33.1%), C18:3 (8.6%), and C16:0 (6.8%). This study demonstrates that nitrogen source can strongly influence lipid production and composition of N. oceanica IMET1.展开更多
Hydroesterification process has been presented biodiesel production from oil the green microalga Nannochloropsis oculata raw materials. Biodiesel studied in this work is the main product got from the hydroesterificati...Hydroesterification process has been presented biodiesel production from oil the green microalga Nannochloropsis oculata raw materials. Biodiesel studied in this work is the main product got from the hydroesterification of biomass the Nannochloropsis oculata and was obtained from esterification of fatty acid (product of a hydrolysis reaction) with methanol. It was used as catalyst the niobic acid pure and supported on δ-aluminum. The product was evaluated by gas chromatography and other analyses. The optimum conditions found in the conversion (%) for the hydrolysis reactions of the biomass (92.3%). Better results were observed in the algae concentration 20%, lead at 300?C with 20% of catalyst. For esterification of fatty acids of Nannochloropsis oculata (92.24%), were observed the molar ratio methanol: fat acid 3, lead at 200°C with 20% of catalyst supported.展开更多
Nannochloropsis oceanica promises to be an industrial-level producer of polyunsaturated fatty acids.In this study,the fastest and slowest growing N.oceanica mutants were selected through N-methyl-N'-nitro-N-nitros...Nannochloropsis oceanica promises to be an industrial-level producer of polyunsaturated fatty acids.In this study,the fastest and slowest growing N.oceanica mutants were selected through N-methyl-N'-nitro-N-nitrosoguanidine mutation,and two mutant strains and the wild type(WT) subjected to transcriptome profiling.It was found that the OD_(680) reads at stationary growth phase of both WT and its mutants were proportional to their cell density,thus indicating their division rate and growth speed during culture.This chemical mutation was effective for improving growth performance,and the fast strain divided faster by upregulating the expression of genes functioning in the cell cycle and downregulating genes involved in synthesis of amino acids,fatty acids,and sugars as well as the construction of ribosome and photosynthetic machinery.However,the relationship among the effected genes responsible for cell cycle,metabolism of fatty and amino acids,and construction of ribosome and photosynthetic machinery remained unclear.Further genetic studies are required for clarifying the genetic/metabolic networks underpinning the growth performance of N.oceanica.These findings demonstrated that this mutation strategy was effective for improving the growth performance of this species and explored a means of microalgal genetic improvement,particularly in species possessing a monoploid nucleus and asexual reproduction.展开更多
Nannochloropsis oculata CS179, a unicellular marine microalga, is rich in long-chain polyunsaturated fatty acids (LCPUFAs). Elongase and desaturase play a key role in the biosynthesis of PUFAs. A new elongase gene, wh...Nannochloropsis oculata CS179, a unicellular marine microalga, is rich in long-chain polyunsaturated fatty acids (LCPUFAs). Elongase and desaturase play a key role in the biosynthesis of PUFAs. A new elongase gene, which encodes 322 amino acids, was identified via RT-PCR and 5' and 3' RACE. The sequence of the elongase gene was blast-searched in the NCBI GenBank and showed a similarity to those of the cryptosporidium. But the NJ-tree revealed that the N. oculata CS179 elongase clustered with those of the microalgae Phaeodactylum tricornutum, Ostreococcus tauri and Thalassiosira pseudonana.展开更多
基金the National Key R&D Program of China(Nos.2018YFD0901506,2018YFD0900305)the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao)(No.2018 SDKJ0406-3)。
文摘Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.
基金Supported by the National Key R&D Program of China(Nos.2018YFD0901506,2018YFD0900305)the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao)(No.2018 SDKJ0406-3)。
文摘Species in genus Nannochloropsis,especially N.oceanica and N.gaditana,have been evolving as the model microalgae for both application and theory studies.The position effect of genome integration,the carrying capability limitation of integrative vectors and the instability of non-integrative vectors have hindered Nannochloropsis genetic modification with concatenate genes and extremely long DNA fragments.The molecular tools including genetic transformation,homologous recombination,gene edition,gene stacking and episome vectors for transient gene expression and diverse reporters and selection markers have been rapidly developing in Nannochloropsis species.The construction of animal and plant artificial chromosomes with“top down”strategy has set fine examples for the construction of Nannochloropsis artificial chromosomes(NannoACs).It seems that the methods and materials to set the foundation for constructing NannoACs are at hands.In this review,we outlined the current status of transgenes in Nannochloropsis species,summarized the limitations of both integrative and non-integrative vectors,and proposed a tentative approach to construct NannoACs by doubling and stabilizing the genome first,and then truncating the natural chromosomes.NannoACs once constructed will facilitate transferring the desired traits and concatenate genes into Nannochloropsis genetic backgrounds,thus contributing towards its genetic improvement and synthetic biological studies.
基金funded by the National Natural Science Foundation of China (No. 31270408)National High Technology Research and Development Program (863 Program) of China (No. 2014AA022001)
文摘Zeocin can cause double strand breaks of DNA and thus is frequently used as a selective antibiotic of eukaryotic Sh ble transformants. In non-transformation system, Zeocin may function as a mutagen if not totally lethal. To verify such function of Zeocin, we mutated Nannochloropsis oceanica by increasing the concentration of Zeocin in medium gradually, and isolated a N. oceanica strain(single cell culture) which survived Zeocin up to 10.0μg mL^(-1). The Zeocin-tolerant strain entered the exponential growth phase later and grew slower than the wild strain. Transcriptome profiling showed that the Zeocin-tolerant N. oceanica strain survived Zeocin mainly by adapting(heritable), rather than acclimating(plastic) to Zeocin. Hence mutating N. oceanica with Zeocin was approved effective. Meanwhile, the physiological characteristics of this Zeocin-tolerant strain were demonstrated. As we proposed, N. oceanica tolerated Zeocin by strengthening its protein degradation and antioxidation. The genes controlling cell division and cellular response to stimuli may also have played important roles in the reduction of growth and the tolerance to Zeocin. Our findings evidenced that Zeocin can serve as an appropriate mutagen of microalgae. Creating variations through mutation with Zeocin may help to study the genetic basis of the traits of this monoploidy and asexual microalga, as well as improve its production.
基金supported by the Basic Scientific Fund for National Public Research Institutes of China (No. 2016Q07)
文摘The marine microalga Nannochloropsis sp.contains various elongases and desaturases that are critical for biosynthesis of polyunsaturated fatty acids.A full-length cDNA encoding a long-chain fatty acid elongase,named Ns FAE,was cloned from Nannochloropsis sp..The open reading frame of Ns FAE(GenBank accession no.MF680548)consisted of 1068 bp and encoded a predicted protein of 355 amino acids with molecular mass 38.8 k Da.The deduced polypeptide showed 43%–44%identity to fatty acyl elongases from other algae.RT-PCR experiments indicated that the Ns FAE gene exhibited the highest expression in Nannochloropsis sp.at 72 h(i.e.,during the third growth stage)and the expression was significantly lower in the other four growth stages.Plasmid p Ns FAE-CRISPR and a recombinant DNA fragment(ADH1p-Ns FAE-CYCt)were transformed into Saccharomyces cerevisiae strain BY4742 using the CRISPR-Cas system.Yeast transformants containing Ns FAE produced three fatty acids not normally present in wild-type BY4742-linoleic acid,linolenic acid and eicosadienoic acid-indicating that Ns FAE encodes a functional elongase enzyme.
基金Supported by the Fundamental Research Funds for the Central Universities(No.201762017)
文摘Marine microalga Nannochloropsis oceanica LAMB0001 were domesticated (~730 generations,~two days each) to adapt freshwater BG11 medium. A number of freshwater medium adapted colony-derived strains were obtained. The strains were verified phylogenetically to be N. oceanica LAMB0001 based on the 18S ribosomal RNA gene. Freshwater-medium adapted strain (FA1) grew faster in the BG11 medium prepared with freshwater than wild-type N. oceanica grew in f/2 medium prepared with seawater. We assumed that (1) the expression patterns of the genes that expressed differentially between FA1 and the wild-type N. oceanica exposing to the BG11 medium (WT-F) have been reprogrammed;(2) the physiological processes in which these genes involved have been modified;and (3) a Gene Ontology (GO) term or a KEGG pathway enriched by DEGs between FA1 and WT-F has been up- or down-regulated if it was enriched simultaneously by up- or down-regulated DEGs between FA1 and WT-F, respectively. Under these assumptions, we found that FA1 reprogrammed the expression patterns of a set of genes that involved in cell adhesion, membrane and membrane integrity, material transportation, cell movement, and cellular signaling network. These changes in cellular functions and metabolic pathways indicate that the microalga modified its gene expression pattern in a wide function range and at a high regulation rank in order to adapt to the freshwater medium. It is feasible to domesticate marine microalgae to a freshwater habitat, which may aid to modify their cultivation performances.
基金financially supported by the Fundamental Research Funds for the Central Universities (No. 201762017)
文摘In this study, the RNA sequencing was used to describe the response of Nannochloropsis oceanica, a marine microalga, to benzo[a]pyrene(BaP), a polycyclic aromatic hydrocarbon, in order to elucidate the metabolic pathways(or processes) involved in microalgal response to this stubborn pollutant. N. oceanica was exposed to BaP at a concentration of 90 μg L^-1 for 72 h, and its transcriptome was sequenced through the Illumina HiSeq^TM 2500 platform. This concentration of BaP was selected as it is the lowest for modeling the most appropriate growth inhibition of N. oceanica for transcriptomic analysis. We found that N. oceanica responds to BaP through degrading proteins and repairing DNA damaged by BaP. In addition, superoxide dismutase(SOD) strengthened its performance by increasing its transcript abundance. The physiological mechanism underlining the response of N. oceanica to BaP as revealed by transcriptomic analysis was consistent with the biochemical insights documented previously.
基金financially supported by the National Key R&D Program of China(Nos.2018YFD0900305 and 2018YFD0901506)the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao)(No.2018SDKJ0406-3)the Fundamental Research Funds for the Central Uni-versities(No.201762017)
文摘In order to decipher the hygromycin B tolerance and resistance mechanisms of Nannochloropsis oceanica,the transcriptome profiles of a transgenic strain carrying a randomly integrated hygromycin B resistant gene,a hygromycin B-adaptive strain and a wild type strain of N.oceanica were compared by transcriptome sequencing(RNA-seq)without referring to a high quality genome sequence.The results showed that the adaptive strain adapts to the hygromycin B existing environments mainly by intensifying the expressions of the efflux pump ABC and MFS superfamily transporter genes,thus reducing the intracellular concentration of hygromycin B.The transgenic strain obtains the hygromycin B resistance ability solely by expressing exogenous resistance gene.Accordingly,the screening and maintenance of N.oceanica transformants should be carried out at an antibiotics concentration higher than the adaptive threshold.Our findings can help the genetic modification of N.oceanica with the application of hygromycin B.
基金the National High Technology Research and Development Program of China (2010AA10A403)the National Natural Science Foundation of China (No.40976076)+1 种基金the Major State Basic Research Development Program of China (973 Program) (2011CB200901)the Basic Research Program of Municipal Bureau of Science and Technology of Qingdao (09-1-3-22-jch)
文摘Nannochloropsis is a genus of marine eukaryotic unicellular algae,which belongs to class Eustigmatophyceae.The spe-cies of Nannochloropsis which are fine rotifer feed and rich in eicosapentaenoic acid(EPA)are economically important.Species in this genus are usually 2-5μm in size and are morphologically similar,which makes their identification difficult.We obtained a monoclone of Nannochloropsis with plating method in this study.DNA was extracted and the quality was determined by restriction enzyme digestion and spectrophotometer analysis.The DNA extracted was used to amplify the sequences of 18S ribosomal RNA gene,ITS region of ribosomal RNA transcription unit and rbcL gene.The phylogenetic analysis was carried out by constructing the neighbor-joining trees with Tamura-Nei distances.The phylogenetic analysis showed that the monoclone is N.oceanica.
基金supported by the National Key Technology R&D Programme (No. 2011BAD14B04)。
文摘This study determined growth and lipid accumulation in Nannochloropsis sp. MASCC 11 cultivated at different pH, temperatures, and CO2 concentrations in 10-day period. The suitability for biodiesel production was also evaluated based on the fatty acid profiles of microalgae lipid. Nannochloropsis sp. MASCC 11 showed an excellent tolerance to acidic pH(as low as 4.0), high temperatures(at least 40℃), and high CO2 concentrations(5%-15%), which are major stressed conditions in flue gas. The highest algal biomass was acquired at pH of 9.0(0.44 g L^-1), a temperature of 35℃(0.63 g L^-1), and a CO2 concentration of 5%(2.27 g L^-1). Maximum lipid production was obtained at p H of 6.0(108.2 mg L^-1), a temperature of 35℃(134.6 mg L^-1), and a CO2 concentration of 5%(782.7 mg L^-1). Synthesis of polyunsaturated fatty acids(PUFAs) in biomass was stimulated under high CO_2 concentrations, remaining above 80% of total fatty acids, mainly composed of C16:3, C18:2, and C18:3. This led to the algae-based biodiesel having a lower oxidation stability, better cold flow properties, and other parameters such as its kinematic viscosity, cetane number, and specific gravity complied with ASTM or EN 14214 biodiesel specifications. Therefore, the improvement of oxidative stability needs to be considered before Nannochloropsis sp. MASCC 11 lipid can be used for biodiesel production, even if this species can grow well under stressful conditions.
基金Supported by the National Key R&D Program of China(Nos.2018YFD0900305,2018YFD0901506)the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao)(No.2018SDKJ0406-3)the Fundamental Research Funds for the Central Universities(No.201762017)。
文摘Nannochloropsis oceanica is a marine microalgal species with both economic value and biological importance.It grows fast,contains rich oils,reproduces asexually,holds a small and haploidy genome,and is easy to be modified genetically.However,the genetic study of N.oceanica is scarce.Very less genetic bases of its traits have been deciphered,and no gene has been isolated from it with the function verified simultaneously via either genetic or reverse genetic approaches or both(de novo cloned).Changing medium salinity may aid to control harmful organisms met during large scale cultivation.As a stress,it may also facilitate the accumulation of desirable chemicals including fatty acids.In order to decipher the genetic basis of the low salinity tolerance of N.oceanica,we mutated N.oceanica with Zeocin.In total,five mutant bulks were constructed at equal number of cells,100 mutants each,which were tolerant to a discontinuous serial of salinities from that of 100%of f/2 to that of a mixture of 4%of f/2 and 94%of BG11.The bulks were genotyped through whole genome re-sequencing and analyzed with bulked mutant analysis(BMA)newly modified from bulked segregant analysis(BSA).In total,47 SNPs and 112 InDels were found to associate with the low salinity tolerance,and around them a set of low salinity tolerance associating genes were identified.A set of annotatable genes commonly found between control and different salinities indicated that the genes functioning in gene expression,energy metabolism and cellular structure may be involved in the low salinity tolerance.These associating molecular markers and genes around them were not enough for outlining the physiological mechanism underlining the tolerance;however they should aid to improve N.oceanica genetically.
文摘The effects of the nitrogen sources sodium nitrate (NaNO3) and urea (CH4N2O) on growth, lipid production, and fatty acid composition of Nannochloropsis oceanica IMET1 were investigated. Nitrogen source affected cell density, dry cell weight, and lipid production. Cells grown in the nitrate medium increased dry cell weight and lipid weight in comparison with cells grown in the urea medium. The composition of fatty acids varied with nitrogen sources. IMET1cultured in the nitrate medium mainly contained C18:2 (14.9%) and C16:0 (6.3%) fatty acids, while IMET1 in the urea medium mainly contained C22:0 (33.1%), C18:3 (8.6%), and C16:0 (6.8%). This study demonstrates that nitrogen source can strongly influence lipid production and composition of N. oceanica IMET1.
文摘Hydroesterification process has been presented biodiesel production from oil the green microalga Nannochloropsis oculata raw materials. Biodiesel studied in this work is the main product got from the hydroesterification of biomass the Nannochloropsis oculata and was obtained from esterification of fatty acid (product of a hydrolysis reaction) with methanol. It was used as catalyst the niobic acid pure and supported on δ-aluminum. The product was evaluated by gas chromatography and other analyses. The optimum conditions found in the conversion (%) for the hydrolysis reactions of the biomass (92.3%). Better results were observed in the algae concentration 20%, lead at 300?C with 20% of catalyst. For esterification of fatty acids of Nannochloropsis oculata (92.24%), were observed the molar ratio methanol: fat acid 3, lead at 200°C with 20% of catalyst supported.
基金Supported by the National Natural Science Foundation of China(No.31270408)the National High Technology Research and Development Program of China(863 Program)(No.2014AA022001)
文摘Nannochloropsis oceanica promises to be an industrial-level producer of polyunsaturated fatty acids.In this study,the fastest and slowest growing N.oceanica mutants were selected through N-methyl-N'-nitro-N-nitrosoguanidine mutation,and two mutant strains and the wild type(WT) subjected to transcriptome profiling.It was found that the OD_(680) reads at stationary growth phase of both WT and its mutants were proportional to their cell density,thus indicating their division rate and growth speed during culture.This chemical mutation was effective for improving growth performance,and the fast strain divided faster by upregulating the expression of genes functioning in the cell cycle and downregulating genes involved in synthesis of amino acids,fatty acids,and sugars as well as the construction of ribosome and photosynthetic machinery.However,the relationship among the effected genes responsible for cell cycle,metabolism of fatty and amino acids,and construction of ribosome and photosynthetic machinery remained unclear.Further genetic studies are required for clarifying the genetic/metabolic networks underpinning the growth performance of N.oceanica.These findings demonstrated that this mutation strategy was effective for improving the growth performance of this species and explored a means of microalgal genetic improvement,particularly in species possessing a monoploid nucleus and asexual reproduction.
基金supported by the Project for Supporting the National Development (No. 2006BAD09A03)National 863 Program (No. 2007AA09Z427)
文摘Nannochloropsis oculata CS179, a unicellular marine microalga, is rich in long-chain polyunsaturated fatty acids (LCPUFAs). Elongase and desaturase play a key role in the biosynthesis of PUFAs. A new elongase gene, which encodes 322 amino acids, was identified via RT-PCR and 5' and 3' RACE. The sequence of the elongase gene was blast-searched in the NCBI GenBank and showed a similarity to those of the cryptosporidium. But the NJ-tree revealed that the N. oculata CS179 elongase clustered with those of the microalgae Phaeodactylum tricornutum, Ostreococcus tauri and Thalassiosira pseudonana.
