Arthrospira platensis is an economically important cyanobacterium; and it has been used widely in food and pharmaceutical industries. The phycocyanin(PC) from A. platensis is extremely valuable in medicine and molecul...Arthrospira platensis is an economically important cyanobacterium; and it has been used widely in food and pharmaceutical industries. The phycocyanin(PC) from A. platensis is extremely valuable in medicine and molecular biology due to its antioxidation and anti-tumoring activity and applicability as fluorescence protein tag. In present study, two recombinant plasmids, one contained the phycocyanobilin(PCB)-producing genes(hox1 and pcyA) while the other contained the phycobiliprotein gene(cpcB) and the lyase gene(either cpcS/U or cpcT), were constructed and synchronically transferred into E. coli in order to test the the activities of relevant lyases for catalysing PCB addition to CpcB during synthesizing fluorescent PC holo-β-subunit(β-PC) of A. platensis. As was evidenced by the fluorescence emitted at a peak specific for PC, CpcB was successfully synthesized in E. coli, to which co-expressed PCBs attached though at a relatively low efficiency. The results showed that the attachment of PCBs to CpcB were carried out mainly by co-expressed CpcS/U but CpcB also showed some autocatalytic activity. Currently, no CpcT activity was detected in this E. coli expression system. Further studies will be conducted to improve the efficiency of fluorescent PC synthesis in E. coli.展开更多
To study the promoter activities of genes cpcT 2 and cpcS 2,several upstream DNA fragments of these two genes with different lengths were selected.These fragments were fused with promoterless gfp（reporter gene） for ...To study the promoter activities of genes cpcT 2 and cpcS 2,several upstream DNA fragments of these two genes with different lengths were selected.These fragments were fused with promoterless gfp（reporter gene） for constructing five recombinant plasmids.Then,these recombinant plasmids were transferred into Nostoc PCC 7120 by conjugation.The promoter activities of genes cpcT 2 and cpcS 2 were determined by observing the fluorescence of green fluorescent protein（GFP） with a fluorescence microscope.The result showed that the 1 300 bp（-1 300 to 0 bp） and 2 600 bp（-2 600 to 0 bp） upstream fragments of cpcT 2 had strong promoter activity and the promoter activity of the 680 bp（-680 to 0 bp） fragment was weaker than that of two above fragments of cpcT 2.The 2 000 bp（-2 000 to 0 bp） upstream fragment of cpcS 2 revealed weak promoter activity.This showed that a strong promoter of cpcT 2 was located in the upstream fragment between-680 and-1 300 bp.展开更多
基金supported by the National Science and Technology Major Project of China (2008ZX08001-004)
文摘Arthrospira platensis is an economically important cyanobacterium; and it has been used widely in food and pharmaceutical industries. The phycocyanin(PC) from A. platensis is extremely valuable in medicine and molecular biology due to its antioxidation and anti-tumoring activity and applicability as fluorescence protein tag. In present study, two recombinant plasmids, one contained the phycocyanobilin(PCB)-producing genes(hox1 and pcyA) while the other contained the phycobiliprotein gene(cpcB) and the lyase gene(either cpcS/U or cpcT), were constructed and synchronically transferred into E. coli in order to test the the activities of relevant lyases for catalysing PCB addition to CpcB during synthesizing fluorescent PC holo-β-subunit(β-PC) of A. platensis. As was evidenced by the fluorescence emitted at a peak specific for PC, CpcB was successfully synthesized in E. coli, to which co-expressed PCBs attached though at a relatively low efficiency. The results showed that the attachment of PCBs to CpcB were carried out mainly by co-expressed CpcS/U but CpcB also showed some autocatalytic activity. Currently, no CpcT activity was detected in this E. coli expression system. Further studies will be conducted to improve the efficiency of fluorescent PC synthesis in E. coli.
基金Supported by the National Natural Science Foundation of China(30870541, 31070743)China Postdoctoral Science Foundation (20100471191)
文摘To study the promoter activities of genes cpcT 2 and cpcS 2,several upstream DNA fragments of these two genes with different lengths were selected.These fragments were fused with promoterless gfp（reporter gene） for constructing five recombinant plasmids.Then,these recombinant plasmids were transferred into Nostoc PCC 7120 by conjugation.The promoter activities of genes cpcT 2 and cpcS 2 were determined by observing the fluorescence of green fluorescent protein（GFP） with a fluorescence microscope.The result showed that the 1 300 bp（-1 300 to 0 bp） and 2 600 bp（-2 600 to 0 bp） upstream fragments of cpcT 2 had strong promoter activity and the promoter activity of the 680 bp（-680 to 0 bp） fragment was weaker than that of two above fragments of cpcT 2.The 2 000 bp（-2 000 to 0 bp） upstream fragment of cpcS 2 revealed weak promoter activity.This showed that a strong promoter of cpcT 2 was located in the upstream fragment between-680 and-1 300 bp.
