Dunaliella salina is a classic halophilic alga.However,its molecular mechanisms in response to high salinity at the post transcriptional level remain unknown.A unique halophilic alga strain,DS-CN1,was screened from fo...Dunaliella salina is a classic halophilic alga.However,its molecular mechanisms in response to high salinity at the post transcriptional level remain unknown.A unique halophilic alga strain,DS-CN1,was screened from four D.salina strains via cell biological,physiological,and biochemical methods.High-throughput sequencing of small RNAs(sRNAs)of DS-CN1 in culture medium containing 3.42-mol/L NaCl(SS group)or 0.05-mol/L NaCl(CO group)was performed on the BGISEQ-500 platform.The annotation and sequences of D.salina sRNAs were profiled.Altogether,44 novel salt stress-responsive microRNAs(miRNAs)with a relatively high C content,with the majority of them being 24 nt in length,were identified and characterized in DS-CN1.Twenty-one differentially expressed miRNAs(DEMs)in SS and CO were screened via bioinformatic analysis.A total of 319 putative salt stress-related genes targeted(104 overlapping genes)by novel miRNAs in this alga were screened based on our previous transcriptome sequencing research.Furthermore,these target genes were classified and enriched by GO and KEGG pathway analysis.Moreover,5 novel DEMs(dsa-mir3,dsa-mir16,dsa-mir17,and dsa-mir26 were significantly upregulated,and dsa-mir40 was significantly downregulated)and their corresponding 10 target genes involved in the 6 significantly enriched metabolic pathways were verified by quantitative real-time PCR.Next,their regulatory relationships were comprehensively analyzed.Lastly,a unique salt stress response metabolic network was constructed based on the novel DEM-target gene pairs.Taken together,our results suggest that 44 novel salt stress-responsive microRNAs were identified,and 4 of them might play important roles in D.salina upon salinity stress and contribute to clarify its distinctive halophilic feature.Our study will shed light on the regulatory mechanisms of salt stress responses.展开更多
[Objective] The aim was to obtain the full-length cDNA sequence of Dunaliella parva β-actin gene.[Method] Based on the highly conserved amino acid regions of known β-actin,a pair of degenerate primers was synthesize...[Objective] The aim was to obtain the full-length cDNA sequence of Dunaliella parva β-actin gene.[Method] Based on the highly conserved amino acid regions of known β-actin,a pair of degenerate primers was synthesized to amplify 533 bp cDNA sequences in Dunaliella parva.Then,the 5＇ genomic DNA and 3＇ cDNA sequences were obtained by Genome walking and 3＇-RACE technology based on the obtained sequence.According to the sequences of the 5＇-termini and 3＇-termini,specific primers were synthesized to obtain the full-length cDNA.[Result] The full-length β-actin cDNA included 1 137 bp open reading frame（ORF）,617 bp of 3＇ noncoding region.Similarity analysis indicated that the highest similarity was found between Dunaliella parva and Dunaliella salina.The Dunaliella parva β-actin also showed wide similarity with other algae.[Conclusion] The full-length cDNA sequence of D.parva was firstly obtained,which was highly conserved.展开更多
Dunaliella salina is known as one of the most salt-tolerant eukaryotic or- ganisms, and the most ideal model organism for studying plant adaption to high salinity. In recent years, the study on molecular mechanism of ...Dunaliella salina is known as one of the most salt-tolerant eukaryotic or- ganisms, and the most ideal model organism for studying plant adaption to high salinity. In recent years, the study on molecular mechanism of salt tolerance in Dunaliella salina has become the focus of scholars at home and abroad with the development of molecular biological techniques. This study reviewed studies on adaption of Dunaliella salina to high salinity in aspects of osmotic adjustment, salt tolerance-related genes and proteins of Dunaliella salina and signal transduction pathway of salt stress.展开更多
Dunaliella salina is an important model organism for investigating the salt tolerance mechanism of plant. MAPK is the key gene in the molecular pathway of salt tolerance of plant. In this experiment, the open reading ...Dunaliella salina is an important model organism for investigating the salt tolerance mechanism of plant. MAPK is the key gene in the molecular pathway of salt tolerance of plant. In this experiment, the open reading frame （ORF） of DsMAPK gene was amplified by PCR. The target fragment was cloned in pGS-21a, a prokaryotic expression vector with GST-tag. The recombinant plasmid pGS-21a- DsMAPK was then transformed into E. coil BL21 （DE3）. The expression was induced with IPTG. Then the expression form of the recombinant protein was analyzed. The expression products were purified with GST-SefinoseTM Kit and identified with SDS-PAGE and Western blot. The results showed the recombinant expression vector pGS-21a-DsMAPK was constructed successfully, and the molecular weight of the expressed recombinant protein was as same as expected. Western blot analysis showed the purified recombinant protein could be identified specially by the anti- GST antibody and had a good immunological activity. The successful expression of DsMAPK will lay a basis for the further research on the role of DsMAPK in the salt tolerance mechanism at the protein level.展开更多
The quality of light is an important abiotic factor that affects the growth and development of photosynthetic organisms.In this study,we exposed the unicellular green alga Dunaliella salina to red(660 nm)and blue(450 ...The quality of light is an important abiotic factor that affects the growth and development of photosynthetic organisms.In this study,we exposed the unicellular green alga Dunaliella salina to red(660 nm)and blue(450 nm)light and analyzed the cell growth,total carotenoid content,and transcriptomes.The growth of D.salina was enhanced by illumination with red light,whereas blue light was not able to promote the algal growth.In contrast,the total carotenoid content increased under both red and blue light.The RNA of D.salina was sequenced and the transcriptomic response of algal cells to red and blue light was investigated.Six transcripts encoding for the blue light receptor cryptochrome were identified,and transcripts involved in the carotenoid metabolism were up-regulated under both red and blue light.Transcripts encoding for photoprotective enzymes related to the scavenging of reactive oxygen species were up-regulated under blue light.The present transcriptomic study provides a more comprehensive understanding of carotenoid biosynthesis in D.salina under different wavelengths of light.展开更多
A Dunaliella strain was isolated from Yuncheng Salt Lake,Shanxi,China(111.05°E,35.03°N).Morphological characteristics and molecular data were used to evaluate the relationship of this algal strain to other D...A Dunaliella strain was isolated from Yuncheng Salt Lake,Shanxi,China(111.05°E,35.03°N).Morphological characteristics and molecular data were used to evaluate the relationship of this algal strain to other Dunaliella展开更多
Protein kinase C (PKC) has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. We previously isolated the full-length PKC gene fro...Protein kinase C (PKC) has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. We previously isolated the full-length PKC gene from Dunaliella salina (DsPKC) using rapid amplification of cDNA ends (RACE) and RT-PCR methods. And we submitted the mRNA sequence of DsPKC gene to NCBI (Genbank No. JN625213). In the present paper, the DsPKC gene open reading frame obtained by PCR was cloned into pGS-21a vector and transformed into Escherichia coli to generate the fusion protein. Bioinformatics analysis revealed that DsPKC gene was a member of serine/threonine kinase with two conserved domains and highly conserved motifs. The DsPKC was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.2 mmol L 1 at 37℃. Under salt stress, the fu- sion protein Green Fluorescent Protein (GFP)-DsPKC was transferred from the cytoplasm to the cell membrane. The expression pat- tern of DsPKC gene was analyzed using real-time quantitative PCR, and indicated that DsPKC gene was up-regulated by 3.0 mol L 1 NaCl at 12 h, which was significantly higher than in control values (P < 0.05). These results suggest that the DsPKC gene plays an important role in response to salt stress in D. salina.展开更多
Algal allelopathy is a manifold ecological/physiological phenomenon that is focused on chemical interactions and autotoxicity. We investigated the allelopathic interactions between Karenia mikimotoi and Dunaliella sal...Algal allelopathy is a manifold ecological/physiological phenomenon that is focused on chemical interactions and autotoxicity. We investigated the allelopathic interactions between Karenia mikimotoi and Dunaliella salina in laboratory cultures based on different temperature （15℃, 20℃, and 25℃） and lighting （40,80, and 160 umol/（m2·s）） conditions. The growth of D. salina in bi-algae culture （1：1 size/density） was significantly restrained. The results of cell-free filtrate culture indicate that direct cell-to- cell contact was not necessary in interspecific competition. Further experimental results demonstrated that allelochemicals released from K. miMmotoi were markedly influenced by both temperature （P=0.013） and irradiance （P=0.003）, resulting in different growth characteristics olD. salina in filtrate mediums. Compared with the plateau period, K. mikimotoi exudates in the exponential phase had a stronger short-term inhibition effect on D. salina in normal conditions. A clear concentration-dependent relationship was observed in the effect of allelochemicals released from K. mikimotoi with low-promoting and high-repressing effects on D. Salina in a short time-scale. In addition, allelopathic substances remain stable and effective under high temperature and pressure stress. Many flocculent sediments adhering with D. salina cells were observed in all filtrate mediums, while the quantity and color depended on the original culture conditions.展开更多
Microalgal lipids are regarded as main future feedstock of biofuels for its higher efficiency of accumulation and sus- tainable production. In order to investigate the effect of various nitrogen to phosphorus ratios o...Microalgal lipids are regarded as main future feedstock of biofuels for its higher efficiency of accumulation and sus- tainable production. In order to investigate the effect of various nitrogen to phosphorus ratios on cells growth, chlorophyll content and accumulation of lipids in Dunaliella tertiolecta, experiments were carried out in modified microalgal medium with inorganic nitrogen （nitrate-nitrogen） or organic nitrogen （urea-nitrogen） as the sole nitrogen source at initial N：P ratios ranging from 1：1 to 32：1. The favorable N：P of 16：1 in the nitrate-N or urea-N medium yielded the maximum cell density and specific growth rate. Decrease in chlorophyll content were observed at the N：P of 4：1 in both nitrate-N and urea-N cultures. It was also observed that the maximum lipids concentration was obtained at the N：P of 4：1 in both nitrate and urea nutrient medium. The lipid productivity and lipid content of cultures in the urea-N medium at the N：P of 4： lwere markedly higher than those from cultures with other N：P ratios （p〈 0.05）. The results of this work illustrate the possibility that higher ratios of nitrogen to phosphorus have enhancing effect on cells growth of D. tertiolecta. Conversely, higher lipid accumulation is associated with a decrease in chlorophyll content under lower ratios of nitro- gen to phosphorus. The results confirm the hypothesis of this study that a larger metabolic flux has been channeled to lipid accumu- lation in D. tertiolecta cells when the ratios of nitrogen to phosphorus drop below a critical level.展开更多
基金Supported by the National Natural Science Foundation of China(No.32170204)Science and Technology Strategy Research Special Project of Shanxi Province of China(No.202204031401051)+2 种基金the Basic Research Programs of Shanxi Province of China(No.202103021224009)the Teaching Reform and Innovation Project of Colleges and Universities in Shanxi of China(No.J20220046)the Shanxi“1331 Project”.
文摘Dunaliella salina is a classic halophilic alga.However,its molecular mechanisms in response to high salinity at the post transcriptional level remain unknown.A unique halophilic alga strain,DS-CN1,was screened from four D.salina strains via cell biological,physiological,and biochemical methods.High-throughput sequencing of small RNAs(sRNAs)of DS-CN1 in culture medium containing 3.42-mol/L NaCl(SS group)or 0.05-mol/L NaCl(CO group)was performed on the BGISEQ-500 platform.The annotation and sequences of D.salina sRNAs were profiled.Altogether,44 novel salt stress-responsive microRNAs(miRNAs)with a relatively high C content,with the majority of them being 24 nt in length,were identified and characterized in DS-CN1.Twenty-one differentially expressed miRNAs(DEMs)in SS and CO were screened via bioinformatic analysis.A total of 319 putative salt stress-related genes targeted(104 overlapping genes)by novel miRNAs in this alga were screened based on our previous transcriptome sequencing research.Furthermore,these target genes were classified and enriched by GO and KEGG pathway analysis.Moreover,5 novel DEMs(dsa-mir3,dsa-mir16,dsa-mir17,and dsa-mir26 were significantly upregulated,and dsa-mir40 was significantly downregulated)and their corresponding 10 target genes involved in the 6 significantly enriched metabolic pathways were verified by quantitative real-time PCR.Next,their regulatory relationships were comprehensively analyzed.Lastly,a unique salt stress response metabolic network was constructed based on the novel DEM-target gene pairs.Taken together,our results suggest that 44 novel salt stress-responsive microRNAs were identified,and 4 of them might play important roles in D.salina upon salinity stress and contribute to clarify its distinctive halophilic feature.Our study will shed light on the regulatory mechanisms of salt stress responses.
基金Supported by Comprehensive Strategic Cooperation Project betweenGuangdong Province and Chinese Academy of Sciences (2010A0-90100010)National Key Technology R&D Program of the 12thFive-year Plan of China (2011BAD14B03)Foundation of KeyLaboratory of Renewable Energy and Natural Gas Hydrate,ChineseAcademy of Sciences (y107j6)~~
文摘[Objective] The aim was to obtain the full-length cDNA sequence of Dunaliella parva β-actin gene.[Method] Based on the highly conserved amino acid regions of known β-actin,a pair of degenerate primers was synthesized to amplify 533 bp cDNA sequences in Dunaliella parva.Then,the 5＇ genomic DNA and 3＇ cDNA sequences were obtained by Genome walking and 3＇-RACE technology based on the obtained sequence.According to the sequences of the 5＇-termini and 3＇-termini,specific primers were synthesized to obtain the full-length cDNA.[Result] The full-length β-actin cDNA included 1 137 bp open reading frame（ORF）,617 bp of 3＇ noncoding region.Similarity analysis indicated that the highest similarity was found between Dunaliella parva and Dunaliella salina.The Dunaliella parva β-actin also showed wide similarity with other algae.[Conclusion] The full-length cDNA sequence of D.parva was firstly obtained,which was highly conserved.
基金Supported by National Natural Science Foundation of China(3147226030972240)~~
文摘Dunaliella salina is known as one of the most salt-tolerant eukaryotic or- ganisms, and the most ideal model organism for studying plant adaption to high salinity. In recent years, the study on molecular mechanism of salt tolerance in Dunaliella salina has become the focus of scholars at home and abroad with the development of molecular biological techniques. This study reviewed studies on adaption of Dunaliella salina to high salinity in aspects of osmotic adjustment, salt tolerance-related genes and proteins of Dunaliella salina and signal transduction pathway of salt stress.
基金Supported by National Natural Science Foundation of China(3147226030972240)~~
文摘Dunaliella salina is an important model organism for investigating the salt tolerance mechanism of plant. MAPK is the key gene in the molecular pathway of salt tolerance of plant. In this experiment, the open reading frame （ORF） of DsMAPK gene was amplified by PCR. The target fragment was cloned in pGS-21a, a prokaryotic expression vector with GST-tag. The recombinant plasmid pGS-21a- DsMAPK was then transformed into E. coil BL21 （DE3）. The expression was induced with IPTG. Then the expression form of the recombinant protein was analyzed. The expression products were purified with GST-SefinoseTM Kit and identified with SDS-PAGE and Western blot. The results showed the recombinant expression vector pGS-21a-DsMAPK was constructed successfully, and the molecular weight of the expressed recombinant protein was as same as expected. Western blot analysis showed the purified recombinant protein could be identified specially by the anti- GST antibody and had a good immunological activity. The successful expression of DsMAPK will lay a basis for the further research on the role of DsMAPK in the salt tolerance mechanism at the protein level.
基金Supported by the National Natural Science Foundation of China(No.41506188)the China Nantong Municipal Applied Basic Research Program(No.MS12017025-2)the Tianjin Demonstration Project for Innovative Development of Marine Economy(No.BHSF2017-21)
文摘The quality of light is an important abiotic factor that affects the growth and development of photosynthetic organisms.In this study,we exposed the unicellular green alga Dunaliella salina to red(660 nm)and blue(450 nm)light and analyzed the cell growth,total carotenoid content,and transcriptomes.The growth of D.salina was enhanced by illumination with red light,whereas blue light was not able to promote the algal growth.In contrast,the total carotenoid content increased under both red and blue light.The RNA of D.salina was sequenced and the transcriptomic response of algal cells to red and blue light was investigated.Six transcripts encoding for the blue light receptor cryptochrome were identified,and transcripts involved in the carotenoid metabolism were up-regulated under both red and blue light.Transcripts encoding for photoprotective enzymes related to the scavenging of reactive oxygen species were up-regulated under blue light.The present transcriptomic study provides a more comprehensive understanding of carotenoid biosynthesis in D.salina under different wavelengths of light.
基金funded by the National Natural Science Foundation of China (Grant No. 31170193 to Shulian XIE)
文摘A Dunaliella strain was isolated from Yuncheng Salt Lake,Shanxi,China(111.05°E,35.03°N).Morphological characteristics and molecular data were used to evaluate the relationship of this algal strain to other Dunaliella
基金the functional analysis of PKC signaling pathway involved in response to salt stress of Dunaliella salinathe National Natural Science Foundation of China (No. 31472260)
文摘Protein kinase C (PKC) has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. We previously isolated the full-length PKC gene from Dunaliella salina (DsPKC) using rapid amplification of cDNA ends (RACE) and RT-PCR methods. And we submitted the mRNA sequence of DsPKC gene to NCBI (Genbank No. JN625213). In the present paper, the DsPKC gene open reading frame obtained by PCR was cloned into pGS-21a vector and transformed into Escherichia coli to generate the fusion protein. Bioinformatics analysis revealed that DsPKC gene was a member of serine/threonine kinase with two conserved domains and highly conserved motifs. The DsPKC was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.2 mmol L 1 at 37℃. Under salt stress, the fu- sion protein Green Fluorescent Protein (GFP)-DsPKC was transferred from the cytoplasm to the cell membrane. The expression pat- tern of DsPKC gene was analyzed using real-time quantitative PCR, and indicated that DsPKC gene was up-regulated by 3.0 mol L 1 NaCl at 12 h, which was significantly higher than in control values (P < 0.05). These results suggest that the DsPKC gene plays an important role in response to salt stress in D. salina.
基金Supported by the State Key Laboratory of Satellite Ocean Environment Dynamics(Second Institute of Oceanography,SOA)(No.SOED1418)the Public Science and Technology Research Funds Projects of Ocean(No.201305027)+1 种基金the National Natural Science Foundation of China(No.91128212)the Research Fund for the Doctoral Program of Higher Education of China(No.20110132120025)
文摘Algal allelopathy is a manifold ecological/physiological phenomenon that is focused on chemical interactions and autotoxicity. We investigated the allelopathic interactions between Karenia mikimotoi and Dunaliella salina in laboratory cultures based on different temperature （15℃, 20℃, and 25℃） and lighting （40,80, and 160 umol/（m2·s）） conditions. The growth of D. salina in bi-algae culture （1：1 size/density） was significantly restrained. The results of cell-free filtrate culture indicate that direct cell-to- cell contact was not necessary in interspecific competition. Further experimental results demonstrated that allelochemicals released from K. miMmotoi were markedly influenced by both temperature （P=0.013） and irradiance （P=0.003）, resulting in different growth characteristics olD. salina in filtrate mediums. Compared with the plateau period, K. mikimotoi exudates in the exponential phase had a stronger short-term inhibition effect on D. salina in normal conditions. A clear concentration-dependent relationship was observed in the effect of allelochemicals released from K. mikimotoi with low-promoting and high-repressing effects on D. Salina in a short time-scale. In addition, allelopathic substances remain stable and effective under high temperature and pressure stress. Many flocculent sediments adhering with D. salina cells were observed in all filtrate mediums, while the quantity and color depended on the original culture conditions.
基金partially supported by Tianjin Marine Revitalization Plan of Science and Technology (No.KJXH2013-16) from Tianjin Oceanic Administration,China
文摘Microalgal lipids are regarded as main future feedstock of biofuels for its higher efficiency of accumulation and sus- tainable production. In order to investigate the effect of various nitrogen to phosphorus ratios on cells growth, chlorophyll content and accumulation of lipids in Dunaliella tertiolecta, experiments were carried out in modified microalgal medium with inorganic nitrogen （nitrate-nitrogen） or organic nitrogen （urea-nitrogen） as the sole nitrogen source at initial N：P ratios ranging from 1：1 to 32：1. The favorable N：P of 16：1 in the nitrate-N or urea-N medium yielded the maximum cell density and specific growth rate. Decrease in chlorophyll content were observed at the N：P of 4：1 in both nitrate-N and urea-N cultures. It was also observed that the maximum lipids concentration was obtained at the N：P of 4：1 in both nitrate and urea nutrient medium. The lipid productivity and lipid content of cultures in the urea-N medium at the N：P of 4： lwere markedly higher than those from cultures with other N：P ratios （p〈 0.05）. The results of this work illustrate the possibility that higher ratios of nitrogen to phosphorus have enhancing effect on cells growth of D. tertiolecta. Conversely, higher lipid accumulation is associated with a decrease in chlorophyll content under lower ratios of nitro- gen to phosphorus. The results confirm the hypothesis of this study that a larger metabolic flux has been channeled to lipid accumu- lation in D. tertiolecta cells when the ratios of nitrogen to phosphorus drop below a critical level.
