Arthrospira (Spirulina) is an economically important filamentous cyanobacterium. As a healthy food it contains high content of proteins, essential amino acids, vitamins, minerals and fatty acids. Among the components it is worthwhile to mention that the light-harvesting pigment c-phycocyanin is very plentiful, which not only plays a great role in photosynthesis, but also can be developed for pharmaceutical usage (Bhat and Madyastha, 2000; Reddy et al. 2003; Subhashini et al. 2004). The studies on the c-phycocyanin from Arthrospira have been mainly focused on the pharmacological function and protein features, less work on the c-phycocyanin gene has been done till the cloning of the coding region sequence of the c-phycocyanin gene from Arthrospira maxima (Yu et al. 2002). To our knowledge, there is no report on the regulatory sequence of the gene. Recently, a 2086bp fragment of Arthrospira platensis FACHB341 has been cloned in our laboratory, including the complete b subunitgene (cpcB) and the 419 bp upstream sequence. In cyanobacteria, few details are known of the mechanisms through which the expression of the c-phycocyanin is regulated. Investigation of the upstream sequence can help to understand the regulation mechanism, which will be valuable to the production of c-phycocyanin.

Studies on the regulatory sequence of a gene usually need to creat a reporting system, and the function of the sequence can be determined from the expression of the reporter gene. Since no stable genetic transformation system has been established in Arthrospira platensis (Toyomizu et al. 2001), structure and function studies of the upstream sequence of the cpcB gene must rely on a heterologous system. In the present paper, Synechococcus sp. strain PCC7942 was applied to construct the reporting system, with the reporter gfp gene integrated in the genome of the alga, and a series of functional analyses of the upstream sequence of cpcB gene were conducted through deletion mutation.

First, the 419bp upstream sequence of cpcB gene was subcloned and fused with gfp gene on the plasmid pEGFP-1. To construct the recombinant alga, the isiAB locus of Synechococcus sp.PCC7942 was selected as the integration platform, a 1.7kb fragment of isiAB was amplified by PCR and ligated with cloning vector pMD18-T, after some modifications to the new plasmid, the GFP expression cassette was inserted in the middle of the isiAB fragment to construct the homologous recombination vector. After transformation the recombinant alga can be selected on the BG11 plate containing 5 µg/ml Kanamycin. The fluorescent microscope analysis showed the upstream sequence of cpcB gene could drive the expression of GFP in Synechococcus sp., and the expression level can be measured quantitatively by flow cytometry.

To investigate whether the upstream sequence of cpcB gene was light-responsive, the recombinant alga was cultured in several different light intensities (1.0, 10.0, 25.0, 40.0 and 60.0 µmol m^-2 s^-1). It showed that in the scope of light intensity larger than 10.0µmol m^-2 s^-1, low light intensity improved the expression of GFP, the lower the light intensity, the higher the GFP level; but light intensity below 10.0 µmol m^-2 s^-1 might lead to a low GFP expression level.

To define the function of different regions on the 419 bp upstream sequence of cpcB gene, a series of 5' end deletion mutants of the upstream sequence of cpcB gene were obtained through site-directed mutagenesis (TaKaRa MutanBest Kit), then the corresponding homologous recombination vectors and recombinant algae were constructed, and the GFP levels of each recombinant alga in low light intensity (10.0 µmol m^-2 s^-1) and in high light intensity (40.0 µmol m^-2 s^-1) were analyzed by flow cytometry. It could be concluded from the results that a light-responsive element was possibly located between -276 and -218, the 85 bp sequence adjacent to the coding region appeared to be the minimum region to keep promoter activity, and there might be some cis elements improving the GFP expression in the regions from -419 to -276, and from -218 to -130, respectively.

BHAT, V.B. and MADYASTHA, K.M. C-phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro. Biochemical and Biophysical Research Communications, August 2000, vol. 275, no. 1, p. 20-25.

REDDY, M.C.; SUBHASHINI, J.; MAHIPAL, S.V.; BHAT, V.B.; SRINIVAS REDDY, P.; KIRANMAI, G.; MADYASTHA, K.M. and REDDANNA, P. C-Phycocyanin, a selective cyclooxygenase-2 inhibitor, induces apoptosis in lipopolysaccharide-stimulated RAW 264.7 macrophages. Biochemical and Biophysical Research Communications, May 2003, vol. 304, no. 2, p.385-392.

SUBHASHINI, J.; MAHIPAL, S.V.; REDDY, M.C.; MALLIKARJUNA REDDY, M.; RACHAMALLU, A. and REDDANNA, P. Molecular mechanisms in C-Phycocyanin induced apoptosis in human chronic myeloid leukaemia cell line-K562. Biochemical Pharmacology, August 2004, vol. 68, no. 3, p. 453-462.

TOYOMIZU, M.; SUZUKI, K.; KAWATA, Y.; KOJIMA, H. and AKIBA, Y. Effective transformation of the cyanobacterium Spirulina platensis using electroporation. Journal of Applied Phycology, June 2001, vol. 13, no. 3, p. 209-214.

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