To produce mouse metallothionein-1 (mMT-I ) in cyanobacterium Anabaena sp. PCC 7120, a novel Escherichia co/i-cyanobacterium shuttle fusion expression vector, pKG-MT, was constructed. Via this vector, mMT-I cDNA which...To produce mouse metallothionein-1 (mMT-I ) in cyanobacterium Anabaena sp. PCC 7120, a novel Escherichia co/i-cyanobacterium shuttle fusion expression vector, pKG-MT, was constructed. Via this vector, mMT-I cDNA which was fused with a carboxyl terminal extension of the 26 kD glutathione-S-trans-ferase (GST) containing a thrombin specific site was expressed in Anabaena under the control of tac promoter. SDS-polyacrylamid gel electrophoresis (SDS-PAGE) showed that the fusion protein GST-MT was expressed in the transgenic Anabaena sp. PCC 7120 after induction with isopropylthio-β-D-galactoside (IPTG). Glutatione-S-transferase metallothionein (GST-MT) was purified from the crude extracts by affinity chromatography on immobilized glutathione and mMT- I was obtained by digesting the fusion protein with thrombin on column and gel filtration on Sephadex G-50. SDS-PAGE demonstrated that the purified mMT- I was the desired protein. The result of ELISA for the purified mMT-I showed that the recovery of mMT- I from the transgenic cyanobacterium was about 0.6 mg/g fresh weight. According to the data of atomic absorption assay, metal-binding activity of the purified mMT-I was almost the same as that of wild type MT.展开更多
The effect of iron deficiency on heterocyst differentiation and some physiological properties of the filamentous cyanobacterium Anabaena sp. PCC 7120 was investigated. Under moderate iron limitation conditions, ac...The effect of iron deficiency on heterocyst differentiation and some physiological properties of the filamentous cyanobacterium Anabaena sp. PCC 7120 was investigated. Under moderate iron limitation conditions, achieved by addition of iron chelator 2,2′\|Dipyridyl (<80 μmol/L) led to delayed heterocyst differentiation, no heterocyst differentiation was observed under severe iron limitation conditions, when the concentration of 2,2′\|Dipyridyl in the medium was more than 100 μmol/L . It seemed that there are certain iron\|regulated genes or operons whose function is to control heterocyst development. In addition, iron deficiency impaired the growth. Low\|iron cells had a decrease in the quantities of pigment content (chlorophyll and phycocyanin content),the whole cell in vivo absorbance spectra confirmed the decrease, the protein electrophoretic profiles revealed that iron\|deficient cells had less protein bands, with the increase of 2,2′\|Dipyridyl ,the protein bands was more and more less. And differently, iron deficiency also caused an increase of ROS (Reactive Oxygen Species)and SOD activity, it suggests that iron deficiency led to oxidative stress, which generally occured under high\|iron conditions.展开更多
Mixotrophic growth is one potential mode for mass culture of microalgae and cyanobacteria particularly suitable for the production of high value bioactive compounds and fine chemicals.The typical heterocystous cyanoba...Mixotrophic growth is one potential mode for mass culture of microalgae and cyanobacteria particularly suitable for the production of high value bioactive compounds and fine chemicals.The typical heterocystous cyanobacterium Anabaena sp.PCC 7120 was grown in the presence of exogenous glucose in light.Glucose improved the cell growth evidently,the maximal specific growth rate under mixotrophic condition（0.38 d 1）being 1.6-fold of that of photoautotrophic growth.Mixotrophy caused a variation in cellular pigment composition,increasing the content of chlorophyll a and decreasing the contents of carotenoid and phycobiliprotein relative to chlorophyll a.Fluorescence emission from photosystem II（PSII）relative to photosystem I was enhanced in mixotrophic cells,implying an increased energy distribution in PSII.Glucokinase（EC 2.7.1.2）activity was further induced in the presence of glucose.The mixotrophic culture was scaled up in a 15 L airlift photobioreactor equipped with an inner and an outer light source.A modified Monod model incorporating the specific growth rate and the average light intensity in the reactor was developed to describe cell growth appropriately.The understanding of mixotrophic growth and relevant physiological features of Anabaena sp.PCC 7120 would be meaningful for cultivation and exploitation of this important cyanobacterial strain.展开更多
ThefecC gene encoding a putative iron (III) dicitrate transporter was cloned from nitrogen-fixing cyanobacteriumAnabaena sp. PCC 7120, and inactivated. The mutant grows normally in medium with NO 3 ? , NH 4 + or witho...ThefecC gene encoding a putative iron (III) dicitrate transporter was cloned from nitrogen-fixing cyanobacteriumAnabaena sp. PCC 7120, and inactivated. The mutant grows normally in medium with NO 3 ? , NH 4 + or without combined nitrogen. But in iron-deficient medium, the mutant grows slowly. Photosynthetic properties were compared between the mutant and the wildtype strain, the content of photosynthetic pigments in the mutant is lower than that of the wild-type. The results of RT-PCR experiments show that thefecC gene is expressed under iron-deficient conditions, but is not expressed under iron-replete conditions. These results revealed thatfecC gene product is required for optimal growth under iron-deficient conditions inAnabaena sp. PCC 7120. Key words Anabaena sp. PCC 7120 - fecC - iron deficiency - photosynthetic properties - expression CLC number Q 933 Foundation item: Supported by the National Natural Sciences Foundation of China (30070154), the Frontier Science Projects Program of the Institute of Hydrobiology, the Chinese Academy of Sciences (220316), State Key Project on Cyanobacterial Bloom Control in Lake Dianchi (K99-05-35-01)Biography: XU Wen-liang (1974-), male, Ph. D, research direction: molecular genetics of cyanobacteria.展开更多
The type Ⅱ toxin-antitoxin genes are responsible for the phenotypic switch to a quasi-dormant state that enables cell survival under stresses,a similar function to heterocyst of cyanobacteria. In this paper,we partic...The type Ⅱ toxin-antitoxin genes are responsible for the phenotypic switch to a quasi-dormant state that enables cell survival under stresses,a similar function to heterocyst of cyanobacteria. In this paper,we particularly study the role of gene pair all3211-asl3212 under Spectinomycin stress to reveal how the type Ⅱ toxin-antitoxin involved in environmental stress responses. Bioinformatics prediction shows that toxin protein gene All3211 is homologous to Maz F,a member of maz EF family that encoding nucleases. We clone gene all3211-asl3212 into expression vectors to identify its molecular characteristics. Deletion mutant strains of all3211-asl3212 are selected in a tri-parental mating screen. Phenotype comparisons of mutant and wild type reveals no difference of single-deletion-mutants in pigment integrity,the sensitivity to antibiotics,and heterocyst formation. The results show that deletion mutation of single TAS gene pair all3211-asl3212 results in limited effects on the cellular growth of PCC 7120. Thus,we suggest that dosage compensating might be provided from redundant genes or bypass pathways to offset obvious phenotypic differences.展开更多
Theconstruction of an integrative shuttle expression vector and potential utility was reported inEscherichiacoliandAnabaena(Nostoc) sp. strain PCC 7120. The vector comprised of the following elements: (a) an intergeni...Theconstruction of an integrative shuttle expression vector and potential utility was reported inEscherichiacoliandAnabaena(Nostoc) sp. strain PCC 7120. The vector comprised of the following elements: (a) an intergenic non-coding region fromAnabaenato facilitate its genomic integration (b) a strong functional PpsbAIpromoter fromAnabaenafor desired gene expression and (c) neomycin phosphotransferase gene with its own promoter for the selection of transfor-mants. The constructed vectorpAnFP was evaluated by cloning, transfer and expression of thegfpgene encoding green fluorescent protein. When theE.coliandAnabaenasp. strain PCC 7120 were transformed, intensive green fluorescence produced by the products of GFP protein was observed. This result indicated that the integrative shuttle vector pAnFP can be promisingly used in genome transformation for expression of heterologous genes inE.coliand microalgae such asAnabaenaandNostocstrains.展开更多
Since pepc gene encoding phosphoenolpyruvate carboxylase （PEPCase） has been cloned from Anabaena sp. PCC 7120 and other cyanobacteria, the effects of pepc gene expression on photosynthesis have not been reported yet...Since pepc gene encoding phosphoenolpyruvate carboxylase （PEPCase） has been cloned from Anabaena sp. PCC 7120 and other cyanobacteria, the effects of pepc gene expression on photosynthesis have not been reported yet. In this study, we constructed mutants containing either upregu-lated （forward） or downregulated （reverse） pepc gene in Anabaena sp. PCC 7120. Results from real‐time quantitative polymerase chain reaction （RT‐qPCR）, Western blot and enzymatic analysis showed that PEPCase activity was signifi-cantly reduced in the reverse mutant compared with the wild type, and that of the forward mutant was obviously increased. Interestingly, the net photosynthesis in both the reverse mutant and the forward mutant were higher than that of the wild type, but dark respiration was decreased only in the reverse mutant. The absorbance changes of P700 upon saturation pulse showed the photosystem I （PSI） activity was inhibited, as reflected by Y（I）, and Y（NA） was elevated, and＆amp;nbsp;dark reduction of P700t was stimulated, indicating enhanced cyclic electron flow （CEF） around PSI in the reverse mutant. Additional y, the reverse mutant photosynthesis was higher than that of the wild type in low temperature, low and high pH, and high salinity, and this implies increased tolerance in the reverse mutant through downregulated pepc gene.展开更多
The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-a) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been...The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-a) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recom-binant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with α-32P labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cyto-toxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.展开更多
文摘To produce mouse metallothionein-1 (mMT-I ) in cyanobacterium Anabaena sp. PCC 7120, a novel Escherichia co/i-cyanobacterium shuttle fusion expression vector, pKG-MT, was constructed. Via this vector, mMT-I cDNA which was fused with a carboxyl terminal extension of the 26 kD glutathione-S-trans-ferase (GST) containing a thrombin specific site was expressed in Anabaena under the control of tac promoter. SDS-polyacrylamid gel electrophoresis (SDS-PAGE) showed that the fusion protein GST-MT was expressed in the transgenic Anabaena sp. PCC 7120 after induction with isopropylthio-β-D-galactoside (IPTG). Glutatione-S-transferase metallothionein (GST-MT) was purified from the crude extracts by affinity chromatography on immobilized glutathione and mMT- I was obtained by digesting the fusion protein with thrombin on column and gel filtration on Sephadex G-50. SDS-PAGE demonstrated that the purified mMT- I was the desired protein. The result of ELISA for the purified mMT-I showed that the recovery of mMT- I from the transgenic cyanobacterium was about 0.6 mg/g fresh weight. According to the data of atomic absorption assay, metal-binding activity of the purified mMT-I was almost the same as that of wild type MT.
文摘The effect of iron deficiency on heterocyst differentiation and some physiological properties of the filamentous cyanobacterium Anabaena sp. PCC 7120 was investigated. Under moderate iron limitation conditions, achieved by addition of iron chelator 2,2′\|Dipyridyl (<80 μmol/L) led to delayed heterocyst differentiation, no heterocyst differentiation was observed under severe iron limitation conditions, when the concentration of 2,2′\|Dipyridyl in the medium was more than 100 μmol/L . It seemed that there are certain iron\|regulated genes or operons whose function is to control heterocyst development. In addition, iron deficiency impaired the growth. Low\|iron cells had a decrease in the quantities of pigment content (chlorophyll and phycocyanin content),the whole cell in vivo absorbance spectra confirmed the decrease, the protein electrophoretic profiles revealed that iron\|deficient cells had less protein bands, with the increase of 2,2′\|Dipyridyl ,the protein bands was more and more less. And differently, iron deficiency also caused an increase of ROS (Reactive Oxygen Species)and SOD activity, it suggests that iron deficiency led to oxidative stress, which generally occured under high\|iron conditions.
基金Supported by a grant from the State Key Laboratory of Biochemical Engineering,Institute of Process Engineering,Chinese Academy of Sciences
文摘Mixotrophic growth is one potential mode for mass culture of microalgae and cyanobacteria particularly suitable for the production of high value bioactive compounds and fine chemicals.The typical heterocystous cyanobacterium Anabaena sp.PCC 7120 was grown in the presence of exogenous glucose in light.Glucose improved the cell growth evidently,the maximal specific growth rate under mixotrophic condition（0.38 d 1）being 1.6-fold of that of photoautotrophic growth.Mixotrophy caused a variation in cellular pigment composition,increasing the content of chlorophyll a and decreasing the contents of carotenoid and phycobiliprotein relative to chlorophyll a.Fluorescence emission from photosystem II（PSII）relative to photosystem I was enhanced in mixotrophic cells,implying an increased energy distribution in PSII.Glucokinase（EC 2.7.1.2）activity was further induced in the presence of glucose.The mixotrophic culture was scaled up in a 15 L airlift photobioreactor equipped with an inner and an outer light source.A modified Monod model incorporating the specific growth rate and the average light intensity in the reactor was developed to describe cell growth appropriately.The understanding of mixotrophic growth and relevant physiological features of Anabaena sp.PCC 7120 would be meaningful for cultivation and exploitation of this important cyanobacterial strain.
文摘ThefecC gene encoding a putative iron (III) dicitrate transporter was cloned from nitrogen-fixing cyanobacteriumAnabaena sp. PCC 7120, and inactivated. The mutant grows normally in medium with NO 3 ? , NH 4 + or without combined nitrogen. But in iron-deficient medium, the mutant grows slowly. Photosynthetic properties were compared between the mutant and the wildtype strain, the content of photosynthetic pigments in the mutant is lower than that of the wild-type. The results of RT-PCR experiments show that thefecC gene is expressed under iron-deficient conditions, but is not expressed under iron-replete conditions. These results revealed thatfecC gene product is required for optimal growth under iron-deficient conditions inAnabaena sp. PCC 7120. Key words Anabaena sp. PCC 7120 - fecC - iron deficiency - photosynthetic properties - expression CLC number Q 933 Foundation item: Supported by the National Natural Sciences Foundation of China (30070154), the Frontier Science Projects Program of the Institute of Hydrobiology, the Chinese Academy of Sciences (220316), State Key Project on Cyanobacterial Bloom Control in Lake Dianchi (K99-05-35-01)Biography: XU Wen-liang (1974-), male, Ph. D, research direction: molecular genetics of cyanobacteria.
基金Supported by the National Natural Science Foundation of China(31001099/C190101)Central University Natural Science Foundation of China(CJSl3003,CJS13004)Key Laboratory of Microbiology and Biotrans Formation Funded Projects of South-Central University for Nationalities(XJS09002)
文摘The type Ⅱ toxin-antitoxin genes are responsible for the phenotypic switch to a quasi-dormant state that enables cell survival under stresses,a similar function to heterocyst of cyanobacteria. In this paper,we particularly study the role of gene pair all3211-asl3212 under Spectinomycin stress to reveal how the type Ⅱ toxin-antitoxin involved in environmental stress responses. Bioinformatics prediction shows that toxin protein gene All3211 is homologous to Maz F,a member of maz EF family that encoding nucleases. We clone gene all3211-asl3212 into expression vectors to identify its molecular characteristics. Deletion mutant strains of all3211-asl3212 are selected in a tri-parental mating screen. Phenotype comparisons of mutant and wild type reveals no difference of single-deletion-mutants in pigment integrity,the sensitivity to antibiotics,and heterocyst formation. The results show that deletion mutation of single TAS gene pair all3211-asl3212 results in limited effects on the cellular growth of PCC 7120. Thus,we suggest that dosage compensating might be provided from redundant genes or bypass pathways to offset obvious phenotypic differences.
文摘为研究鱼腥藻PCC7120(Anabaena sp.)基因asr0757/alr0758在毒素-抗毒素系统中的相关生物学功能,设计了特异性引物,扩增目的片段asr0757和alr0758,将目的基因与p MD18-T载体连接构建克隆载体,并对其进行XhoⅠ和NdeⅠ双酶切,再与表达载体p ET-28a连接构建表达重组菌,重组菌的体外表达在IPTG的诱导下进行。经琼脂糖电泳检测,结果扩增出了大小为210 bp的asr0757和342 bp的alr0758目的基因。经SDS-PAGE电泳检测,表达出相对分子质量分别为8.068 k D和12.534 k D的蛋白质。根据结果可初步认定alr0758为毒素基因,asr0757为抗毒素基因,共同构成鱼腥藻PCC7120毒素-抗毒素系统。
文摘Theconstruction of an integrative shuttle expression vector and potential utility was reported inEscherichiacoliandAnabaena(Nostoc) sp. strain PCC 7120. The vector comprised of the following elements: (a) an intergenic non-coding region fromAnabaenato facilitate its genomic integration (b) a strong functional PpsbAIpromoter fromAnabaenafor desired gene expression and (c) neomycin phosphotransferase gene with its own promoter for the selection of transfor-mants. The constructed vectorpAnFP was evaluated by cloning, transfer and expression of thegfpgene encoding green fluorescent protein. When theE.coliandAnabaenasp. strain PCC 7120 were transformed, intensive green fluorescence produced by the products of GFP protein was observed. This result indicated that the integrative shuttle vector pAnFP can be promisingly used in genome transformation for expression of heterologous genes inE.coliand microalgae such asAnabaenaandNostocstrains.
基金supported by the National High-tech R&D Program (863 Program) (Nos.2009AA064401 and 2014AA093506)National Administration Project from Chinese Ministry of Environmental Protection (No.04)supported by Shanghai Universities First-class Disciplines Project of Fisheries
文摘Since pepc gene encoding phosphoenolpyruvate carboxylase （PEPCase） has been cloned from Anabaena sp. PCC 7120 and other cyanobacteria, the effects of pepc gene expression on photosynthesis have not been reported yet. In this study, we constructed mutants containing either upregu-lated （forward） or downregulated （reverse） pepc gene in Anabaena sp. PCC 7120. Results from real‐time quantitative polymerase chain reaction （RT‐qPCR）, Western blot and enzymatic analysis showed that PEPCase activity was signifi-cantly reduced in the reverse mutant compared with the wild type, and that of the forward mutant was obviously increased. Interestingly, the net photosynthesis in both the reverse mutant and the forward mutant were higher than that of the wild type, but dark respiration was decreased only in the reverse mutant. The absorbance changes of P700 upon saturation pulse showed the photosystem I （PSI） activity was inhibited, as reflected by Y（I）, and Y（NA） was elevated, and＆amp;nbsp;dark reduction of P700t was stimulated, indicating enhanced cyclic electron flow （CEF） around PSI in the reverse mutant. Additional y, the reverse mutant photosynthesis was higher than that of the wild type in low temperature, low and high pH, and high salinity, and this implies increased tolerance in the reverse mutant through downregulated pepc gene.
基金Project supported by the National Natural Science Foundation of China (Grant No. 39280016).
文摘The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-a) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recom-binant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with α-32P labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cyto-toxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.
