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
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 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
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 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.
