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
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
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
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.
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.
