Cloning And Expression Of CPCE-F Genes In Spirulina And Its Phylogenetic Analysis

Spirulina is a high economic value prokaryote whose exploitation has a bright future. It is usually argued about the classification. Due to the connection of its nourishment and medical value, the classification of Spirulina appares to be very important. The conventional classification of it is base on the phenotype, but the phenotype is influenced by the surrounding, therefore the difficult appears. By the usage of molecular technique, its classification develops rapidly, and the results of that are obvious. Biliproteins are a wide spread group of brilliantly coloured photoreceptors characterized by linear tetrapyrrolic chromophores, bilins, which are covalently bound to the apoproteins via relatively stable thioether bonds. It is catalyzed by the lyase. The cpcE and cpcF lyase genes are found in the cyanobacterium Synechococcus sp. PCC 7002. The function of them has been proved. We find that the cpcE and cpcF genes have homology in different cyanobacterias. They are fit for phyletic evolution analysis. At present we have not found the new report about phyletic evolution analysis by this genes. We also have not found the new report about the catalytic reaction by this gene in Spirulina itself.The complete sequence of lyase genes were obtained by PCR. Primers were designed according to the published gene sequences of cpcE-F. The sequences are also totally 1488bp. We firstly obtain the sequences of cpcE-F in Spirulina platensis and Spirulina subsalsa. The GC contents of cpcE-F are 50.6% and 50.5%. The similarities of nucleotide in FACHB314 are different in 95.0% and 94.8%, amino acid are different in 93.7% and 93.3%, The similarities of nucleotide in FACHB351 are different in 95.0% and 94.8%, amino acid are different in 94.7% and 95.1%. Mw are 119587.0kDa and 119576.0kDa, pI are 5.76 and 5.69, the aliphatic index are 23.12 and 22.92. We can reach the conclusion that the lyase gene should be use to make a difference in Spirulina.To verify the function of lyase, we have overproduced CpcE and CpcF in Escherichia coli. In vitro, these proteins catalyze the attachment of phycocyanobilin to theα-subunit of apophycocyanin at the appropriate site,α-Cys-84, to form the correct adduct. We also firstly suggest that CpcE and CpcF of Spirulina could catalyse the connection of PCB to phycocyaninαsubunit in vivo, and therefore obtained the transformation activity of light energy.